Interaction of moraxella catarrhalis with epithelial cells, extracellular matrix proteins and the complement system

ABSTRACT

The present invention relates to surface proteins of  Moraxella catarrhalis  and their ability to interact with epithelial cells via cell-associated fibronectin and laminin, and also to their ability to inhibit the complement system. These surface proteins are useful in the preparation of vaccines. The present invention also provides peptides interacting with fibronectin, laminin and the complement system.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to Moraxella catarrhalis and their abilityto interact with epithelial cells via extracellular matrix proteins suchas fibronectin and laminin, and also to their ability to inhibit thecomplement system. The interaction with these extracellular proteins isuseful in the preparation of vaccines.

BACKGROUND ART

The ability to bind epithelial cells is of great importance for severalbacterial species. For example, Staphylococcus aureus and Streptococcuspyogenes possess fibronectin binding proteins (FnBP) with relatedsequence organization. These FnBP are known as Microbial SurfaceComponents Recognizing Adhesive Matrix Molecules (MSCRAMMs).

They exploit the modular structure of fibronectin forming extendedtandem beta-zippers in its binding to fibronectin.[27, 39, 47, 73] Thefunction is to mediate bacterial adhesion and invasion of host cells.

The important mucosal pathogen Moraxella catarrhalis is the thirdleading bacterial cause of acute otitis media in children afterStreptococcus pneumoniae and Haemophilus influenzae.[14, 40, 55] M.catarrhalis is also one of the most common inhabitants of the pharynx ofhealthy children. Furthermore, M. catarrhalis is also a common cause ofsinusitis and lower respiratory tract infections in adults with chronicobstructive pulmonary disease (COPD). [74] The success of this speciesin patients with COPD is probably related in part to its largerepertoire of adhesins.

Recent years focus of research has been on the outer membrane proteinsand their interactions with the human host.[6, 48, 56] Some of theseouter membrane proteins appear to have adhesive functions includingamongst others, M. catarrhalis IgD binding protein (MID, also designatedHag), protein CD, M. catarrhalis adherence protein (McaP) and theubiquitous surface proteins (Usp).[1, 22, 33, 48, 61, 81, 84]

SUMMARY OF THE INVENTION

In view of the fact that M. catarrhalis has been found to be such aleading cause of infections in the upper and lower airways, there is acurrent need to develop vaccines which can be used against M.catarrhalis.

The aim of the present invention has therefore been to find out in whichway M. catarrhalis interacts with epithelial cells in the body andaffects the immune system. In this way, substances that can act asvaccines against M. catarrhalis can be developed.

In this study, using M. catarrhalis mutants derived from clinicalisolates, the inventors have been able to show that both UspA1and A2bind fibronectin and laminin. Furthermore, the inventors have been ableto show that M. catarrhalis interfere with the classical pathway of thecomplement system, and also to elucidate in which way they interfere.

Many bacteria adhere to epithelial cells via fibronectin bindingMSCRAMMS.[54, 77] Pseudomonas aeruginosa has a FnBP that binds tocellular associated fibronectin on nasal epithelial cells.[69] Blockingthe bacteria-fibronectin protein interactions may help the host tissueto overcome the infection. In fact, it has been shown that antibodiesagainst a S. aureus FnBP resulted in rapid clearance of the bacteria ininfected mice.[71]

Recombinant truncated UspA1/A2 proteins together with smaller fragmentsspanning the entire molecule have been tested according to the presentinvention for fibronectin binding. Both UspA1 and A2 bound fibronectinand the fibronectin binding domains were found to be located withinUspA1²⁹⁹⁻⁴⁵² and UspA2¹⁶⁵⁻³¹⁸. These two truncated proteins bothinhibited binding of M. catarrhalis to Chang conjunctival epithelialcells to a similar extent as anti-fibronectin antibodies. Theobservations made show that both M. catarrhalis UspA1 and A2 areinvolved in the adherence to epithelial cells via cell-associatedfibronectin. The biologically active sites within UspA1²⁹⁹⁻⁴⁵² andUspA2¹⁶⁵-318 are therefore suggested as potential candidates to beincluded in a vaccine against M. catarrhalis.

Further, the inventors have studied and characterized binding of M.catarrhalis to laminin. M. catarrhalis is a common cause of infectiousexacerbations in patients with COPD. The success of this species inpatients with COPD is probably related in part to its large repertoireof adhesins. In addition, there are pathological changes such as loss ofepithelial integrity with exposure of basement membrane where thelaminin layer itself is thickened in smokers.[4] Some pathogens havebeen shown to be able to bind laminin and this may contribute to theirability to adhere to such damaged and denuded mucosal surfaces. Theseinclude pathogens known to cause significant disease in the airways suchas S. aureus and P. aeruginosa amongst others.[7, 63] The presentinventors have been able to show that M. catarrhalis ubiquitous surfaceprotein (Usp) A1 and A2 also bind to laminin. Laminin binding domains ofUspA1 and A2 were, amongst others, found within the N-terminal halves ofUspA1⁵⁰⁻⁴⁹¹ and UspA2³⁰⁻³⁵¹. These domains are also containing thefibronectin binding domains. However, the smallest fragments that boundfibronectin, UspA1²⁹⁹⁻⁴⁵² and UspA2¹⁶⁵⁻³¹⁸, did not bind laminin to anyappreciable extent. Fragments smaller than the N-terminal half of UspA1(UspA1⁵⁰⁻⁴⁹¹) lose all its laminin binding ability, whereas with UspA2,only UspA2³⁰⁻¹⁷⁰ bound laminin albeit at a lower level than the wholerecombinant protein (UspA2³⁰⁻⁵³⁹). These findings suggest that differentparts of the molecule might have different functional roles. UspA1⁵⁰⁻⁷⁷⁰was also found to have laminin binding properties.

Comparing the smallest laminin binding regions of UspA1 and A2, we findthat there is, however, little similarity by way of amino acid homologybetween UspA2³⁰⁻¹⁷⁰ and UspA1⁵⁰⁻⁴⁹¹ (data not shown). This is notsurprising as it is a known fact that both proteins have a‘lollipop’-shaped globular head structure despite having only 22%identity in both N terminal halves.[2, 32]

The biologically active sites within UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ aresuggested as potential candidates to be included in a vaccine against M.catarrhalis.

Finally, the inventors have studied the interaction between M.catarrhalis ubiquitous surface proteins A1 and A2 and the innate immunesystem, and have found that M. catarrhalis interferes with thecomplement system. The complement system is one of the first lines ofinnate defence against pathogenic microorganisms, and activation of thissystem leads to a cascade of protein deposition on the bacterial surfaceresulting in formation of the membrane attack complex or opsonization ofthe pathogen followed by phagocytosis. [85, 86] One of the mostimportant complement proteins is C3, which is present in the circulationin a concentration similar to some immunoglobulins (1-1.2 mg/ml). C3does not only play a crucial role as an opsonin, but also is the commonlink between the classical, lectin and alternative pathways of thecomplement activation. The alternative pathway functions asamplification loop for the classical and lectin pathways and can also bespontaneously activated by covalent attachment of C3 to the surface of amicrobe in the absence of complement inhibitors. C3 deposition requiresthe presence of an internal thioester bond, formed in the native proteinby the proximity of a sulfhydryl group (Cys¹⁰¹⁰) and a glutamyl carbonyl(Gln¹⁰¹²) on the C3 α-chain.[76] Proteolytic cleavage of a 77-residuepeptide from the amino terminus of the C3 α-chain generates C3a(anaphylatoxin) and C3b. Attachment of C3b is then accomplished througha covalent link between the carbonyl group of the metastable thioesterand either —NH2 or —OH groups of proteins or carbohydrate structures onthe activator surface. [36, 37] M. catarrhalis UspA1 and A2 have beenfound to non-covalently and in a dose dependent manner bind both thethird component of complement (C3) from EDTA-treated serum andmethylamine treated C3 (C3met). UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ have beenfound to bind to C3 and C3met. The C3-binding region for UspA2 was foundto mainly be localised in UspA2²⁰⁰⁻⁴⁵⁸. UspA1 has however been found tohave a minor role in the interactions. The biologically active siteswithin UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ are suggested as potential candidatesto be included in a vaccine against M. catarrhalis.

The UspA family consists of UspA1 (molecular weight 88 kDa), UspA2 (62kDa), and the hybrid protein UspA2H (92 kDa).[2, 43] These proteinsmigrate as high molecular mass complexes in SDS-PAGE, are relativelyconserved and hence important vaccine candidates. The amino acidsequences of UspA1 and A2 are 43% identical and have 140 amino acidresidues that are 93% identical. [2] In a series of 108 M. catarrhalisnasopharyngeal isolates from young children with otitis media, uspA1 anduspA2 genes were detected in 107 (99%) and 108 (100%) of the isolates,respectively. Twenty-one percent were identified as having the hybridvariant gene uspA2H.[50] Moreover, it is known that naturally acquiredantibodies to UspA1 and A2 are bactericidal.[15]

Several functions have been attributed to the UspA family of proteins.UspA1 expression is essential for the attachment of M. catarrhalis toChang conjunctival epithelial cells and Hep-2 laryngeal epithelialcells.[43, 49] In a more recent study, UspA1 was shown to bindcarcinoembryonic antigen related cell adhesion molecules (CEACAM)expressed in the lung epithelial cell line A549.[31] Purified UspA1 hasalso been shown to bind fibronectin in dot blot experiments whilepurified UspA2 did not.[49] Both UspA1 and A2 may play important rolesfor M. catarrhalis serum resistance.[1, 5, 58, 60]

The present invention demonstrates that both UspA1 and A2 aredeterminants for M. catarrhalis binding to fibronectin and laminin inthe clinical isolates M. catarrhalis BBH18 and RH4. Interestingly,recombinant UspA1 and A2 derived from M. catarrhalis Bc5 both boundfibronectin to the same extent. The binding domains for fibronectin werefound within amino acid residues 299 to 452 of UspA1 and 165 to 318 ofUspA2. These two domains share 31 amino acid residues sequence identity.Importantly, truncated protein fragments containing these residues inUspA1 and UspA2 were able to inhibit M. catarrhalis binding to Changepithelial cells suggesting that the interactions with these cells werevia cell-associated fibronectin.

The binding domains for laminin were found within the amino acidresidues mentioned above. Binding assays with recombinant proteinsrevealed that the major binding regions were localized in the N-terminalparts, where both proteins form a globular head.

Bacterial factors mediating adherence to tissue and extracellular matrix(ECM) components are grouped together in a single family named“microbial surface components recognizing adhesive matrix molecules”(MSCRAMMS). Since

UspA1/A2both bind fibronectin and laminin, these proteins can bedesignated MSCRAMMS.

According to one aspect the present invention provides a peptide havingsequence ID no. 1, and fragments, homologues, functional equivalents,derivatives, degenerate or hydroxylation, sulphonation or glycosylationproducts and other secondary processing products thereof.

According to another aspect the present invention provides a peptidehaving sequence ID no. 2, and fragments, homologues, functionalequivalents, derivatives, degenerate or hydroxylation, sulphonation orglycosylation products and other secondary processing products thereof.

According to a further aspect the present invention provides a peptidehaving sequence ID no. 3, and fragments, homologues, functionalequivalents, derivatives, degenerate or hydroxylation, sulphonation orglycosylation products and other secondary processing products thereof.

According to another aspect the present invention provides a peptidehaving sequence ID no. 4, and fragments, homologues, functionalequivalents, derivatives, degenerate or hydroxylation, sulphonation orglycosylation products and other secondary processing products thereof.

According to a further aspect the present invention provides a peptidehaving sequence ID no. 5, and fragments, homologues, functionalequivalents, derivatives, degenerate or hydroxylation, sulphonation orglycosylation products and other secondary processing products thereof.

According to a further aspect the present invention provides a peptidehaving sequence ID no. 6, and fragments, homologues, functionalequivalents, derivatives, degenerate or hydroxylation, sulphonation orglycosylation products and other secondary processing products thereof.

According to another aspect the present invention provides a peptidehaving sequence ID no. 7, and fragments, homologues, functionalequivalents, derivatives, degenerate or hydroxylation, sulphonation orglycosylation products and other secondary processing products thereof.

According to another aspect the present invention provides a peptidehaving sequence ID no. 8, and fragments, homologues, functionalequivalents, derivatives, degenerate or hydroxylation, sulphonation orglycosylation products and other secondary processing products thereof.

According to another aspect the present invention provides a peptidehaving sequence ID no. 9, and fragments, homologues, functionalequivalents, derivatives, degenerate or hydroxylation, sulphonation orglycosylation products and other secondary processing products thereof.

According to another aspect the present invention provides a peptidehaving sequence ID no. 10, and fragments, homologues, functionalequivalents, derivatives, degenerate or hydroxylation, sulphonation orglycosylation products and other secondary processing products thereof.

According to another aspect, the present invention provides use of atleast one peptide according to the invention for the production of amedicament for the treatment or prophylaxis of an infection, preferablyan infection caused by M. catarrhalis, in particular caused by carriageof M. catarrhalis on mucosal surfaces.

According to another aspect, the invention further provides a ligandcomprising a fibronectin binding domain, said ligand consisting of anamino acid sequence selected from the group consisting of Sequence IDNo. 1, Sequence ID No. 2 and Sequence ID No. 3, and fragments,homologues, functional equivalents, derivatives, degenerate orhydroxylation, sulphonation or glycosylation products and othersecondary processing products thereof.

The invention further provides a ligand comprising a laminin bindingdomain, said ligand consisting of an amino acid sequence selected fromthe group consisting of Sequence ID No. 4 to Sequence ID No. 8, andfragments, homologues, functional equivalents, derivatives, degenerateor hydroxylation, sulphonation or glycosylation products and othersecondary processing products thereof.

Further, the present invention provides a ligand comprising a C3 orC3met binding domain, said ligand consisting of an amino acid sequenceselected from the group consisting of Sequence ID No. 4, Sequence ID No.6, Sequence ID No. 9 and Sequence ID No. 10, and fragments, homologues,functional equivalents, derivatives, degenerate or hydroxylation,sulphonation or glycosylation products and other secondary processingproducts thereof.

Further, the present invention provides a medicament comprising one ormore ligands according to the invention and one or more pharmaceuticallyacceptable adjuvants, vehicles, excipients, binders, carriers, orpreservatives.

The present invention further provides a vaccine comprising one or moreligands according to the present invention and one or morepharmaceutically acceptable adjuvants, vehicles, excipients, binders,carriers, or preservatives.

The present invention also provides a method of treating or preventingan infection in an individual, preferably an infection caused by M.catarrhalis, in particular caused by carriage of M. catarrhalis onmucosal surfaces, comprising administering a pharmaceutically effectiveamount of a medicament or vaccine according to the present invention.

Finally, the present invention also provides a nucleic acid sequenceencoding a ligand, protein or peptide of the present invention, as wellas homologues, polymorphisms, degenerates and splice variants thereof.

Further disclosure of the objects, problems, solutions and features ofthe present invention will be apparent from the following detaileddescription of the invention with reference to the drawings and theappended claims.

The expression ligand as it is used herein is intended to denote boththe whole molecule which binds to the receptor and any part thereofwhich includes the receptor binding domain such that it retains thereceptor binding property. Ligands comprising equivalent receptorbinding domains are also included in the present invention.

The expressions fragment, homologue, functional equivalent andderivative relate to variants, modifications and/or parts of thepeptides and protein fragments according to the invention which retainthe desired fibronectin, laminin, C3 or C3met binding properties.

A homologue of UspA1 according to the present invention is defined as asequence having at least 72% sequence identity, as can be seen fromtable 1 below.

A fragment according to the present invention is defined as any of thehomologue sequences which are truncated or extended by 1, 2, 5, 10, 15,20 amino acids at the N-terminus and/or truncated or extended by 1, 2,5, 10, 15, 20 amino acids at the C-terminus.

The expressions degenerate, hydroxylation, sulphonation andglycosylation products or other secondary processing products relate tovariants and/or modifications of the peptides and protein fragmentsaccording to the invention which have been altered compared to theoriginal peptide or protein fragment by degeneration, hydroxylation,sulphonation or glycosylation but which retain the desired fibronectin,laminin, C3 or C3met binding properties.

The present invention concerns especially infections caused by Moraxellacatarrhalis. A peptide according to the present invention can be usedfor the treatment or prophylaxis of otitis media, sinusitis or lowerrespiratory tract infections.

TABLE 1 Multiple alignment of full length UspA1 protein sequences,associated identity percentages O12E O35E O46E P44 TTA24 TTA37 V1171ATCC25238 81 75 83 83 84 79 84 O12E 74 77 83 76 72 75 O35E 72 74 83 7378 O46E 81 81 82 80 P44 81 75 77 TTA24 76 84 TTA37 78

TABLE 2 UspA2 Pileup Analysis - Strains and sequences used acc Straindes sl □TREMBL:O54407_MORCA O54407 O35E Ubiquitous surface 576 protein A2. □TREMBL:Q58XP4_MORCA Q58XP4 MC317 UspA2. 650 □TREMBL:Q848S1_MORCAQ848S1 E22 Ubiquitous surface 877 protein A2H. □TREMBL:Q848S2_MORCAQ848S2 V1122 Ubiquitous surface 616 protein A2. □TREMBL:Q8GH86_MORCAQ8GH86 P44 UspA2. 668 □TREMBL:Q9L961_MORCA Q9L961 TTA37 USPA2H. 889□TREMBL:Q9L962_MORCA Q9L962 O46E USPA2H. 894 □TREMBL:Q9L963_MORCA Q9L963O12E USPA2 (Ubiquitous 684 surface protein A2). □TREMBL:Q9XD51_MORCAQ9XD51 V1171 UspA2. 674 □TREMBL:Q9XD53_MORCA Q9XD53 TTA24 UspA2. 613TREMBL:Q8RTB2_MORCA Q8RTB2 SP12-5 UspA2 686 □TREMBL:Q9XD55_MORCA Q9XD55ATCC25238 UspA2. 630 Forsgren_UspA2 UspA2. 630

Accordingly, the present invention provides a ligand isolated fromMoraxella catarrhalis outer membrane protein which has laminin and/orfibronectin and/or C3-binding, wherein said ligand is a polypeptidecomprising or consisting of an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1-10 which are derived from thefull-length Moraxella catarrhalis BC5 UspA1 & UspA2 sequences shownbelow, or a fragment, homologue, functional equivalent, derivative,degenerate or hydroxylation, sulphonation or glycosylation product orother secondary processing product thereof.

Full-length UspA1 from Moraxella catarrhalis strain BC5:

MNKIYKVKKN AAGHLVACSE FAKGHTKKAV LGSLLIVGIL GMATTASAQK VGKATNKISGGDNNTANGTY LTIGGGDYNK TKGRYSTIGG GLFNEATNEY STIGSGGYNK AKGRYSTIGGGGYNEATNQY STIGGGDNNT AKGRYSTIGG GGYNEATIEN STVGGGGYNQ AKGRNSTVAGGYNNEATGTD STIAGGRKNQ ATGKGSFAAG IDNKANADNA VALGNKNTIE GENSVAIGSNNTVKKGQQNV FILGSNTDTT NAQNGSVLLG HNTAGKAATI VNSAEVGGLS LTGFAGASKTGNGTVSVGKK GKERQIVHVG AGEISDTSTD AVNGSQLHVL ATVVAQNKAD IKDLDDEVGLLGEEINSLEG EIFNNQDAIA KNQADIKTLE SNVEEGLLDL SGRLLDQKAD IDNNINNIYELAQQQDQHSS DIKTLKNNVE EGLLDLSGRL IDQKADLTKD IKALESNVEE GLLDLSGRLIDQKADIAKNQ ADIAQNQTDI QDLAAYNELQ DAYAKQQTEA IDALNKASSA NTDRIATAELGIAENKKDAQ IAKAQANENK DGIAKNQADI QLHDKKITNL GILHSMVARA VGNNTQGVATNKADIAKNQA DIANNIKNIY ELAQQQDQHS SDIKTLAKVS AANTDRIAKN KAEADASFETLTKNQNTLIE QGEALVEQNK AINQELEGFA AHADVQDKQI LQNQADITTN KTAIEQNINRTVANGFEIEK NKAGIATNKQ ELILQNDRLN RINETNNHQD QKIDQLGYAL KEQGQHFNNRISAVERQTAG GIANAIAIAT LPSPSRAGEH HVLFGSGYHN GQAAVSLGAA GLSDTGKSTYKIGLSWSDAG GLSGGVGGSY RWK

Full-length UspA2 from Moraxella catarrhalis strain BC5:

MKTMKLLPLK IAVTSAMIIG LGAASTANAQ AKNDITLEDL PYLIKKIDQN ELEADIGDITALEKYLALSQ YGNILALEEL NKALEELDED VGWNQNDIAN LEDDVETLTK NQNAFAEQGEAIKEDLQGLA DFVEGQEGKI LQNETSIKKN TQRNLVNGFE IEKNKDAIAK NNESIEDLYDFGHEVAESIG EIHAHNEAQN ETLKGLITNS IENTNNITKN KADIQALENN VVEELFNLSGRLIDQKADID NNINNIYELA QQQDQHSSDI KTLKKNVEEG LLELSDHIID QKTDIAQNQANIQDLATYNE LQDQYAQKQT EAIDALNKAS SENTQNIEDL AAYNELQDAY AKQQTEAIDALNKASSENTQ NIEDLAAYNE LQDAYAKQQA EAIDALNKAS SENTQNIAKN QADIANNITNIYELAQQQDK HRSDIKTLAK TSAANTDRIA KNKADDDASF ETLTKNQNTL IEKDKEHDKLITANKTAIDA NKASADTKFA ATADAFTKNG NAITKNAKSI TDLGTKVDGF DSRVTALDTKVNAFDGRITA LDSKVENGMA AQAALSGLFQ PYSVGKFNAT AALGGYGSKS AVAIGAGYRVNPNLAFKAGA AINTSGNKKG SYNIGVNYEF

In a preferred embodiment, the ligand is a polypeptide [or polypeptidetruncate compared with a wild-type polypeptide] comprising or consistingof an amino acid sequence selected from the group consisting of SEQ IDNO: 1-10, or a fragment, homologue, functional equivalent, derivative,degenerate or hydroxylation, sulphonation or glycosylation product orother secondary processing product thereof.

The term ligand is used herein to denote both the whole molecule whichbinds to laminin and/or fibronectin and/or C3 and any part thereof whichincludes a laminin and/or fibronectin and/or C3-binding domain such thatit retains the respective binding property. Thus “ligand” encompassesmolecules which consist only of the laminin and/or fibronectin and/orC3-binding domain i.e. the peptide region or regions required forbinding.

For the purposes of this invention laminin, fibronectin or C3-bindingproperties of a polypeptide can be ascertained as follows:

For the purposes of this invention laminin, fibronectin or C3-bindingproperties of a polypeptide can be ascertained as follows: Polypeptidescan be labelled with ¹²⁵Iodine or other radioactive compounds and testedfor binding in radio immunoassays (RIA) as fluid or solid phase (e.g.,dot blots). Moreover, polypeptides can be analysed for binding withenzyme-linked immunosorbent assays (ELISA) or flow cytometry usingappropriate antibodies and detection systems. Interactions betweenpolypeptides and laminin, fibronectin, or C3 can further be examined bysurface plasmon resonance (Biacore). Examples of methods are exemplifiedin detail in the Material and Methods section.

In another preferred embodiment, the polypeptide [or polypeptidetruncate compared with a wild-type polypeptide] comprises or consists ofat least one of the conserved sequences from within SEQ ID NO: 1-10which are identified in the alignment shown herein. Hence, in thisembodiment, the polypeptide [or polypeptide truncate compared with awild-type polypeptide] comprises of consists of at least one of:

From UspA1 (conserved fragments from the fibronectin binding domain—‘/’separating alternative choices of an amino acid at a position)

G T/V V S V G S/K Q/E/K/A G/N K/N/G/H/S E R Q I V N/H V G A G Q/N/E/K IS/R A/D T/D S T D A V N G S Q L H/Y A L A S/K/T T/A/V I/V S T D A V N GS Q L L L N/ID L S G R L L/I D Q K A D I D N N I N N/H I Y E/D L A Q Q QD Q H S S D I K T L K D Q K A D I D N N I N L A Q Q Q D Q H S S D I K TL K

From UspA2 (conserved fragments from the fibronectin binding domain—‘/’separating alternative choices of an amino acid at a position)

K A D I D N N I N N/H I Y E L A Q Q Q D Q H S S D I K/Q T/A L K/E K/N/SN V/I S/V E G/E L L/F S/N L S D/G H/R T/L I D Q K T/A D I/L A/T Q/K N/D

From UspA2 (conserved fragments from the C3-binding domain—‘/’separating alternative choices of an amino acid at a position)

I E/Q D L A A Y N E L Q D A Y A K Q Q A/T E A I D A L N K A S S E N T QN I A K N Q A D I A N N I T/N N I Y E L A Q Q Q D K/Q H R/S S D I K T LA K T/A S A A N T D/N R I D L A A Y N E L Q D A Y A K Q Q E A I D A L NK A S S E N T Q N I A K N Q A D I A N N I

It will be understood that the polypeptide ligands of the invention cancomprise a laminin and/or fibronectin and/or C3-binding domain ofsequence recited herein which is modified by the addition or deletion ofamino acid residues to or from the sequences recited herein at either orboth the N or C termini, which modified peptides retain the ability tobind laminin and/or fibronectin and/or C3, respectively. Accordingly,the invention further provides a ligand comprising or consisting of apolypeptide in which 50, 40, 30, 20, 10, 5, 3 or 1 amino acid residueshave been added to or deleted from an amino acid sequence recited hereinat either or both the N or C termini, wherein said modified polypeptideretains the ability to bind laminin and/or fibronectin and/or C3; and/orelicit an immune response against the non-modified peptide. By extensionit is meant lengthening the sequence using the context of the peptidefrom the full-length amino acid sequence from which it is derived.

As regards fragments of the polypeptides of the invention, any sizefragment may be used in the invention (based on the homologuesequences/conserved regions/functional domatins discussed herein)provided that the fragment retains the ability to bind laminin and/orfibronectin and/or C3. It may be desirable to isolate a minimal peptidewhich contains only those regions required for receptor binding.

Polypeptide ligands according to the invention may be derived from knownMoraxella catarrhalis UspA1 or UspA2 proteins by truncation at either orboth of the N- and C-termini. Truncates are not the full-length nativeUspA1 or

A2 molecules. Accordingly, the invention further provides a wild-typeUspA1 sequence lacking at least (or exactly) 20, 30, 40, 50, 60, 70, 80,100, 120, 140, 160 etc to 298 amino acids from the N-terminus, and/orlacking at least (or exactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140,160, 180, 200 etc to 450 amino acids from the C-terminus. Preferably,the truncate retains fibronectin binding function (optionally alsolaminin and/or C3-binding).

TABLE 3 Possible combinations of truncations to the N- and C-termini ofwild-type UspA1 protein. No. of amino acids lacking, at least orexactly: From the N- terminus From the C-terminus 0 X 20 30 40 50 60 7080 100 120 140 160 180 200 220 20 0 20 30 40 50 60 70 80 100 120 140 160180 200 220 30 0 20 30 40 50 60 70 80 100 120 140 160 180 200 220 40 020 30 40 50 60 70 80 100 120 140 160 180 200 220 50 0 20 30 40 50 60 7060 100 120 140 160 180 200 220 60 0 20 30 40 50 60 70 80 100 120 140 160180 200 220 70 0 20 30 40 50 60 70 80 100 120 140 160 180 200 220 80 020 30 40 50 60 70 80 100 120 140 160 180 200 220 100 0 20 30 40 50 60 7080 100 120 140 160 180 200 220 120 0 20 30 40 50 60 70 80 100 120 140160 180 200 220 140 0 20 30 40 50 60 70 80 100 120 140 160 180 200 220160 0 20 30 40 50 60 70 80 100 120 140 160 180 200 220 180 0 20 30 40 5060 70 80 100 120 140 160 180 200 220 200 0 20 30 40 50 60 70 80 100 120140 160 180 200 220 220 0 20 30 40 50 60 70 80 100 120 140 160 180 200220 240 0 20 30 40 50 60 70 80 100 120 140 160 180 200 220 260 0 20 3040 50 60 70 80 100 120 140 160 180 200 220 280 0 20 30 40 50 60 70 80100 120 140 160 180 200 220 298 0 20 30 40 50 60 70 80 100 120 140 160180 200 220 From the N- terminus From the C-terminus 0 240 260 280 300320 340 360 380 400 420 440 450 20 240 260 280 300 320 240 360 380 400420 440 450 30 240 260 280 300 320 240 360 380 400 420 440 450 40 240260 280 300 320 240 360 380 400 420 440 450 50 240 260 280 300 320 240360 380 400 420 440 450 60 240 260 280 300 320 240 360 380 400 420 440450 70 240 260 280 300 320 240 360 380 400 420 440 450 80 240 260 280300 320 240 360 380 400 420 440 450 100 240 260 280 300 320 240 360 380400 420 440 450 120 240 260 280 300 320 240 360 380 400 420 440 450 140240 260 280 300 320 240 360 380 400 420 440 450 160 240 260 280 300 320240 360 380 400 420 440 450 180 240 260 280 300 320 240 360 380 400 420440 450 200 240 260 280 300 320 240 360 380 400 420 440 450 220 240 260280 300 320 240 360 380 400 420 440 450 240 240 260 280 300 320 240 360380 400 420 440 450 260 240 260 280 300 320 240 360 380 400 420 440 450280 240 260 280 300 320 240 360 380 400 420 440 450 298 240 260 280 300320 240 360 380 400 420 440 450

Accordingly the invention further provides a wild-type UspA2 sequencelacking at least (or exactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140,160, 164 amino acids from the N-terminus, and/or lacking at least (orexactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 180, 200 etc to 312amino acids from the C-terminus. Preferably, the truncate retainsfibronectin binding function (optionally also laminin and/orC3-binding). Possible truncates may be selected from those shown in thefollowing table, all of which are within the scope of the invention.

TABLE 4 Possible combinations of truncations to the N- and C-termini ofwild-type UspA2 protein No. of amino acids lacking, at least or exactlyFrom the N- terminus From the C-terminus 0 X 20 30 40 50 60 70 80 100120 140 160 180 200 220 240 260 280 300 312 20 0 20 30 40 50 60 70 80100 120 140 160 180 200 220 240 260 280 300 312 30 0 20 30 40 50 60 7080 100 120 140 160 180 200 220 240 260 280 300 312 40 0 20 30 40 50 6070 80 100 120 140 160 180 200 220 240 260 280 300 312 50 0 20 30 40 5060 70 80 100 120 140 160 180 200 220 240 260 280 300 312 60 0 20 30 4050 60 70 80 100 120 140 160 180 200 220 240 260 280 300 312 70 0 20 3040 50 60 70 80 100 120 140 160 180 200 220 240 260 280 300 312 80 0 2030 40 50 60 70 80 100 120 140 160 180 200 220 240 260 280 300 312 100 020 30 40 50 60 70 80 100 120 140 160 180 200 220 240 260 280 300 312 1200 20 30 40 50 60 70 80 100 120 140 160 180 200 220 240 260 280 300 312140 0 20 30 40 50 60 70 80 100 120 140 160 180 200 220 240 260 280 300312 160 0 20 30 40 50 60 70 80 100 120 140 160 180 200 220 240 260 280300 312 164 0 20 30 40 50 60 70 80 100 120 140 160 180 200 220 240 260280 300 312

Accordingly the invention further provides a wild-type UspA2 sequencelacking at least (or exactly) 5, 10, 15, 20, 25 or 29 amino acids fromthe N-terminus, and/or lacking at least (or exactly) 20, 30, 40, 50, 60,70, 80, 100, 120, 140, 160, 180, 200 etc to 453 amino acids from theC-terminus. Preferably, the truncate retains laminin binding function(optionally also fibronectin and/or C3-binding). Possible truncates maybe selected from those shown in the following table, all of which arewithin the scope of the invention.

TABLE 5 Possible combinations of truncations to the N- and C-termini ofwild-type UspA2 protein No. of amino acids lacking, at least or exactly:From the C- terminus From the N-terminus 0 X 5 10 15 20 25 29 20 0 5 1015 20 25 29 30 0 5 10 15 20 25 29 40 0 5 10 15 20 25 29 50 0 5 10 15 2025 29 60 0 5 10 15 20 25 29 70 0 5 10 15 20 25 29 80 0 5 10 15 20 25 29100 0 5 10 15 20 25 29 120 0 5 10 15 20 25 29 140 0 5 10 15 20 25 29 1600 5 10 15 20 25 29 180 0 5 10 15 20 25 29 200 0 5 10 15 20 25 29 220 0 510 15 20 25 29 240 0 5 10 15 20 25 29 260 0 5 10 15 20 25 29 280 0 5 1015 20 25 29 300 0 5 10 15 20 25 29 320 0 5 10 15 20 25 29 340 0 5 10 1520 25 29 360 0 5 10 15 20 25 29 380 0 5 10 15 20 25 29 400 0 5 10 15 2025 29 420 0 5 10 15 20 25 29 440 0 5 10 15 20 25 29 453 0 5 10 15 20 2529

Accordingly the invention further provides a wild-type UspA2 sequencelacking (or exactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160 etc.to 301 amino acids from the N-terminus, and/or lacking at least (orexactly) 20, 30, 40, 50, 60, 70, 80, 100, 120, 140, 160 or 172 aminoacids from the C-terminus. Preferably, the truncate retains C3 bindingfunction (optionally also fibronectin and/or laminin binding). Possibletruncates may be selected from those shown in the following table, allof which are within the scope of the invention.

TABLE 6 Possible combinations of truncations to the N- and C-termini ofwild-type UspA2 protein No. of amino acids lacking, at least or exactly:From the N- terminus From the C-terminus 0 X 20 30 40 50 60 70 80 100120 140 160 172 20 0 20 30 40 50 60 70 80 100 120 140 160 172 30 0 20 3040 50 60 70 80 100 120 140 160 172 40 0 20 30 40 50 60 70 80 100 120 140160 172 50 0 20 30 40 50 60 70 80 100 120 140 160 172 60 0 20 30 40 5060 70 80 100 120 140 160 172 70 0 20 30 40 50 60 70 80 100 120 140 160172 80 0 20 30 40 50 60 70 80 100 120 140 160 172 100 0 20 30 40 50 6070 80 100 120 140 160 172 120 0 20 30 40 50 60 70 80 100 120 140 160 172140 0 20 30 40 50 60 70 80 100 120 140 160 172 160 0 20 30 40 50 60 7080 100 120 140 160 172 180 0 20 30 40 50 60 70 80 100 120 140 160 172200 0 20 30 40 50 60 70 80 100 120 140 160 172 220 0 20 30 40 50 60 7080 100 120 140 160 172 240 0 20 30 40 50 60 70 80 100 120 140 160 172260 0 20 30 40 50 60 70 80 100 120 140 160 172 280 0 20 30 40 50 60 7080 100 120 140 160 172 290 0 20 30 40 50 60 70 80 100 120 140 160 172301 0 20 30 40 50 60 70 80 100 120 140 160 172

Known wild-type UspA1 sequences that may be truncated in this way arethose of strains ATCC25238 (MX2; GenBank accession no. AAD43465), P44(AAN84895), O35E (AAB96359), TTA37 (AAF40122), O12E (AAF40118), O46E(AAF36416), V1171 (AAD43469), TTA24 (AAD43467) (see Table 1/FIG. 19); orBC5 (see above). Known wild-type UspA2 sequences that may be truncatedin this way are those of strains 035E (GenBank accession no. O4407),MC317 (GenBank accession no. Q58XP4), E22 (GenBank accession no.Q848S1), V1122 (GenBank accession no. Q848S2), P44 (GenBank accessionno. Q8GH86) , TTA37 (GenBank accession no. Q9L961), O46E (GenBankaccession no. Q9L962), O12E (GenBank accession no. Q9L963), V1171(GenBank accession no. Q9XD51), TTA24 (GenBank accession no. Q9XD53),SP12-5 (GenBank accession no. Q8RTB2), ATCC25238 (GenBank accession no.Q9XD55) (see Table 2/FIG. 20); or BC5 [Forsgren_UspA2] (see above).

Ideally the UspA1 or UspA2 truncate of this embodiment comprises orconsists of an amino acid sequence selected from the group consisting ofSEQ ID NO: 1-10 or a fragment, homologue, functional equivalent,derivative, degenerate or hydroxylation, sulphonation or glycosylationproduct or other secondary processing product thereof; or comprises orconsists of at least one of the conserved sequences from within theseregions which are identified in the alignment shown in herein, forexample:

From UspA1 (conserved fragments from the fibronectin binding domain—‘/’separating alternative choices of an amino acid at a position)

G T/V V S V G S/K Q/E/K/A G/N K/N/G/H/S E R Q I V N/H V G A G Q/N/E/K IS/R A/D T/D S T D A V N G S Q L H/Y A L A S/K/T T/A/V I/V S T D A V N GS Q L L L N/D L S G R L L/I D Q K A D I D N N I N N/H I Y E/D L A Q Q QD Q H S S D I K T L K D Q K A D I D N N I N L A Q Q Q D Q H S S D I K TL K

From UspA2 (conserved fragments from the fibronectin binding domain—‘/’separating alternative choices of an amino acid at a position)

K A D T D N N I N N/H I Y E L A Q Q Q D Q H S S D I K/Q T/A L K/E K/N/SN V/I E/V E G/E L L/F E/N L S D/G H/R I/L I D Q K T/A D I/L A/T Q/K N/D

From UspA2 (conserved fragments from the C3-binding domain—‘/’separating alternative choices of an amino acid at a position)

I E/Q D L A A Y N E L Q D A Y A K Q Q A/T E A I D A L N K A S S E N T QN I A K N Q A D I A N N I T/N N I Y E L A Q Q Q D K/Q H R/S S D I K T LA K T/A S A A N T D/N R I D L A A Y N E L Q D A Y A K Q Q E A I D A L NK A S S E N T Q N I A K N Q A D I A N N I

It may be convenient to produce fusion proteins containing polypeptideligands as described herein. Accordingly, in a further embodiment, theinvention provides fusion proteins comprising polypeptide ligandsaccording to the invention. Preferably a fusion protein according tothis embodiment is less than 50% identical to any known fully lengthsequence over its entire length. Such fusions can constitute aderivative of the polypeptides of the invention. Further derivatives canbe the use of the polypeptides of the invention to as a carrier tocovalently couple peptide or saccharide moieties. They may be coupledfor instance to pneumococcal capsular oligosaccharides orpolysaccharides, or Moraxella catarrhalis lipooligosaccaharides, ornon-typeable Haemophilus influenzae lipooligosaccaharides.

Homologous peptides of the invention may be identified by sequencecomparison. Homologous peptides are preferably at least 60% identical,more preferably at least 70%, 80%, 90%, 95% or 99% identical inascending order of preference to the peptide sequence disclosed hereinor fragments thereof or truncates of the invention over their entirelength. Preferably the homologous peptide retains the ability to bindfibronectin and/or laminin and/or C3; and/or elicit an immune responseagainst the peptide sequences disclosed herein or fragment thereof.

FIGS. 19 and 20 show an alignment of peptide sequences of UspA1 andUspA2 of different origin which indicates regions of sequence that arecapable of being modified to form homologous sequences whilst retainedfunction (i.e. fibronectin and/or laminin and/or C3 binding ability).Homologous peptides to the BC5 SEQ ID NO: 1-10 peptides are for instancethose sequences corresponding to the BC5 sequence from other strains inFIGS. 19 and 20.

Vaccines of the Invention

The polypeptides/peptides/functionaldomains/homologues/fragments/truncates/derivatives of the inventionshould ideally be formulated as a vaccine comprising an effective amountof said component(s) and a pharmaceutically acceptable excipient.

The vaccines of the invention can be used for administration to apatient for the prevention or treatment of Moraxella catarrhalisinfection or otitis media or sinusitis or lower respiratory tractinfections. They may be administered in any known way, includingintramuscularly, parenternally, mucosally and intranasally.

Combination Vaccines of the Invention

The vaccines of the present invention may be combined with otherMoraxella catarrhalis antigens for prevention or treatment of theaforementioned diseases.

The present inventors have found in particular that Moraxellacatarrhalis has at least 2 means of hampering the host immune systemfrom attacking the organism. In addition to the interaction with C3 (andC4BP) mentioned in the Examples below, M. catarrhalis has a strongaffinity for soluble and membrane bound human IgD through protein MID(also known as OMP106). Moraxella-dependent IgD-binding to B lymphocytesresults in a polyclonal immunoglobulin synthesis which may prohibitproduction of specific monoclonal anti-moraxella antibodies. The factthat M. catarrhalis hampers the human immune system in several waysmight explain why M. catarrhalis is such a common inhabitant of therespiratory tract.

The inventors believe that the combination of antigens involved in theIgD-binding function (MID) and C3-binding function (UspA1 and/or UspA2)can provide an immunogenic composition giving the host enhanceddefensive capabilities against Moraxella's hampering of the human immunesystem thus providing an enhanced decrease in M. catarrhalis carriage onmucosal surfaces.

A further aspect of the invention is therefore a vaccine compositioncomprising an effective amount of UspA1 and/or UspA2 (particularly thelatter) (for instance full-length polypeptides orpolypeptides/peptides/functionaldomains/homologues/fragments/truncates/derivatives of the invention asdescribed herein, preferably which retains a C3-binding function) incombination with an effective amount of protein MID (for instancefull-length polypeptides or polypeptides/peptides/functionaldomains/homologues/fragments/truncates/derivatives thereof, preferablywhich retain a human IgD-binding function), and a pharmaceuticallyacceptable excipient.

Protein MID, and IgD-binding homologous/fragments/truncates thereof isdescribed in WO 03/004651 (incorporated by reference herein).Particularly suitable fragments for this purpose is a polypeptidecomprising (or consisting of) the F2 fragment described in WO 03/004651,or sequences with at least 60, 70, 80, 90, 95, 99% identity theretowhich preferably retain human IgD-binding activity.

The MID and UspA components of this combination vaccine may be separatefrom each other, or may be conveniently fused together by knownmolecular biology techniques.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows thirteen M. catarrhalis strains tested for fibronectinbinding (A). Strong fibronectin binding correlated to UspA1/A2expression as detected by anti-UspA1/A2 pAb (B-I). Flow cytometryprofiles of M. catarrhalis BBH18 wild type and UspA1/A2 deficientmutants show an UspA1/A2-dependent binding to soluble fibronectin. Theprofiles of wild type clinical isolate (B and F) and correspondingmutants devoid of UspA1 (C and G), or UspA2 (D and H), and doublemutants (E and I) lacking both UspA1 and UspA2 are shown. Bacteria wereincubated with rabbit anti-UspA1/A2 or fibronectin followed by ananti-fibronectin pAb. FITC-conjugated rabbit pAb was subsequently addedfollowed by flow cytometry analysis. A typical experiment out of threewith the mean fluorescence intensity (MFI) for each profile is shown.

FIG. 2 shows that M. catarrhalis RH4 UspA2 deficient mutants do not bind¹²⁵I-labeled fibronectin. E. coli BL21 was included as a negativecontrol not binding fibronectin. Bacteria were incubated with¹²⁵I-labeled fibronectin followed by several washes and analyzed in agamma counter. Fibronectin binding to the RH4 wild type expressing bothUspA1 and A2 was set as 100%. The mean values of three independentexperiments are shown. Error bars represent standard deviations (SD).Similar results were obtained with M. catarrhalis BBH18.

FIG. 3 shows pictures that verify that M. catarrhalis mutants devoid ofUspA1 and UspA2 do not bind to immobilized fibronectin. M. catarrhaliswild type was able to adhere at a high density on fibronectin coatedglass slides (A). M. catarrhalis ΔuspA1 mutant was also retained at ahigh density (B), whereas M. catarrhalis ΔuspA2 and ΔuspA1/A2 doublemutants adhered poorly (C and D). Glass slides were coated withfibronectin and incubated with M. catarrhalis RH4 and its correspondingUspA1/A2 mutants. After several washes, bacteria were Gram stained.

FIG. 4 is a graph showing that recombinant UspA1 and A2 bind tofibronectin in a dose-dependent manner. Specific fibronectin binding isshown for UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹. Both UspA proteins (40 nM) werecoated on microtiter plates and incubated with increasing concentrationsof fibronectin followed by detection with rabbit anti-human fibronectinpAb and HRP-conjugated anti-rabbit pAb. Mean values of three separateexperiments are shown and error bars indicate SD.

FIG. 5. The active fibronectin binding domains for UspA1 and UspA2 arelocated between amino acids 299 to 452 and 165 to 318, respectively.Truncated proteins derived from UspA1 (A) and UspA2 (B) are shown. Allfragments were tested for fibronectin binding in ELISA. Forty nM of eachtruncated fragment was coated on microtiter plates and incubated with 80μg/ml and 120 μg/ml fibronectin for UspA1 and UspA2, respectively. Boundfibronectin was detected with rabbit anti-fibronectin pAb followed byHRP-conjugated anti-rabbit pAb. Results are representative for threesets of experiments. Error bars represent SD.

FIG. 6 shows the sequence according to sequence ID No. 1, and thesequence homology between UspA1²⁹⁹⁻⁴⁵² and UspA2¹⁶⁵⁻³¹⁸. The 31identical amino acid residues are within brackets.

FIG. 7 shows that truncated UspA1⁵⁰⁻⁴⁹¹ and UspA1²⁹⁹⁻⁴⁵² fragmentscompetitively inhibit M. catarrhalis UspA-dependent fibronectin binding.M. catarrhalis ΔuspA1/A2 double mutants, which do not bind fibronectin,were included as negative controls. UspA1 recombinant proteins werepre-incubated with 2 mg/100 ml fibronectin before incubation with M.catarrhalis. The mean fluorescence values (MFI) of M. catarrhalis withbound fibronectin detected by FITC conjugated anti-fibronectin pAb inflow cytometry are shown. UspA1⁵⁰⁻⁴⁹¹ and UspA1²⁹⁹⁻⁴⁵² resulted in 95%and 63% inhibition respectively. Error bars represent mean ±SD of threeindependent experiments.

FIG. 8 shows that UspA1²⁹⁹⁻⁴⁵² and UspA2¹⁶⁵⁻³¹⁸ inhibit M. catarrhalisadherence to Chang conjunctival cells via cell-associated fibronectin.Chang epithelial cells expressed fibronectin on the surface as revealedby an anti-fibronectin pAb and flow cytometry (A). Pre-incubation withthe fibronectin binding proteins UspA1²⁹⁹⁻⁴⁵² UspA2¹⁶⁵⁻³¹⁸ oranti-fibronectin pAb resulted in significantly reduced binding by M.catarrhalis RH4 as compared to control recombinant proteins(UspA1⁴³³⁻⁵⁸⁰ and UspA2³⁰⁻¹⁷⁷) and a control antibody (anti-ICAM1 mAb)(B). P<0.05 by two-tailed paired Student's t test. Mean values of threeseparate experiments are shown and error bars indicate SD.

FIG. 9A shows binding of M. catarrhalis RH4 to laminin via UspA1 and A2.M. catarrhalis RH4 wild type (wt) strongly bound to immobilized lamininwith a mean OD of 1.27. RH4ΔuspA1 showed mean OD of 1.14 (89.8% of thewild type). RH4ΔuspA2 and the double mutant RH4ΔuspA1/A2 had a mean ODof 0.19 and 0.23 respectively (15.0% and 18.1% of the wild type). Thiswas not significantly different from the residual adhesion to bovineserum albumin coated plates. Thirty μg/m1 of laminin or bovine serumalbumin were coated on microtiter plates. They were blocked followed byincubation with bacteria suspension and finally washed. Bound bacteriawas detected with anti-MID pAb and HRP-conjugated anti-rabbit pAb. Themean results of 3 representative experiments are shown. Error barsrepresent standard deviations (SD).

FIG. 9B shows the binding of recombinant UspA1 and A2 laminin in adose-dependent manner. Specific laminin binding is shown for UspA1⁵⁰⁻⁷⁷⁰and UspA2³⁰⁻⁵³⁹. Both UspA proteins (40 nM) were coated on microtiterplates and incubated with increasing concentrations of laminin followedby detection with rabbit anti-laminin pAb and HRP-conjugated anti-rabbitpAb. Mean values of three separate experiments are shown and error barsindicate SD.

FIGS. 10A and B show that the active laminin binding domains forUspA1⁵⁰⁻⁷⁷⁰ (A) and UspA2³⁰⁻⁵³⁹ (B) are located in the N-terminalhalves. Forty nM of recombinant UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ togetherwith the truncated proteins were coated on microtiter plates andincubated with 20 μg/ml of laminin followed by detection with rabbitanti-laminin pAb and HRP-conjugated anti-rabbit pAb. Mean values ofthree separate experiments are shown and error bars indicate SD.

FIG. 11 is a schematic illustration of C3, covalent bound C3b and C3met.(A) The C3-molecule in serum consists of one α-chain and one β-chain.(B) The α-chain contains an internal thioester site that afteractivation can attach covalently to a microbial surface. (C) The C3 hasbeen treated with methylamine, which becomes covalently attached to thethioester.

FIG. 12 illustrates that M. catarrhalis counteracts the classical andalternative pathways of the complement system by the outer membraneproteins UspA1 and A2. (A) M. catarrhalis RH4 wild-type (wt), theΔuspA1, the ΔuspA2 or the ΔuspA1/A2 mutants were incubated in thepresence of 10% NHS. (B) The ΔuspA1/A2 mutant was incubated with 10% NHSsupplemented with either EDTA or Mg-EGTA. Bacteria were collected at theindicated time points. After overnight incubation, colony forming units(cfu) were counted. The number of bacteria at the initiation of theexperiments was defined as 100%. Mean values of three separateexperiments are shown and error bars indicate S.D. (A) The mean valuesafter 5 min for the ΔuspA1, the ΔuspA2 or the ΔuspA1/A2 mutants weresignificantly different from the wild-type (P<0.05). (B) The mean valuesafter 5 min for the ΔuspA1/A2 mutant and after 10 min for the ΔuspA1/A2mutant incubated Mg-EGTA were significantly different from the wild-type(P<0.05).

FIG. 13 illustrates that Moraxella catarrhalis binds C3 in serumindependently of complement activation. Flow cytometry profiles showingC3 binding to (A) M. catarrhalis RH4 or (B) Streptococcus pneumoniae.Bacteria were incubated with NHS or NHS pretreated with EDTA.Thereafter, a rabbit anti-human C3d pAb and as a secondary layer aFITC-conjugated goat anti-rabbit pAb were added followed by flowcytometry analysis. Bacteria in the absence of NHS, but in the presenceof both pAb, were defined as background fluorescence. One representativeexperiment out of three is shown.

FIG. 14 illustrates that M. catarrhalis non-covalently binds purifiedmethylamine-treated C3 in a dose-dependent manner, and that the bindingis based on ionic interactions. Flow cytometry profiles showing (A)binding with increasing concentrations of C3met. (B) The meanfluorescence intensity (mfi) of each profile in panel (A) is shown. (C)C3met binding of RH4 decreases with increasing concentrations of NaCl.Bacteria were incubated with C3met with or without NaCl as indicated.C3met binding was measured by flow cytometry as described in FIG. 3.Error bars indicate SD. * P≦0.05, ** P≦0.01, *** P≦0.001.

FIG. 15 illustrates that flow cytometry profiles of M. catarrhalis RH4wild type and UspA1/A2 deficient mutants show a UspA1/UspA2-dependentC3met/C3 binding. The profiles of a wild type clinical isolate (A, F, K)and corresponding mutants devoid of protein MID (B, G, L), UspA1 (C, H,M), UspA2 (D, I, N), or both UspA1 and UspA2 (E, J, O) are shown.Bacteria were incubated with C3met (A-E), NHS-EDTA (F-J) or NHS (K-O)and detected as outlined in FIG. 3. One typical experiment out of threewith the mean fluorescence intensity (mfi) for each profile is shown.

FIG. 16 illustrates that C3met binds to purified recombinantUspA2³⁰⁻⁵³⁹, whereas only a weak C3met binding to UspA1⁵⁰⁻⁷⁷⁰ isobserved. Furthermore, the C3met binding region of UspA2 was determinedto be located between the amino acid residues 200 to 458. (A) Therecombinant UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ were immobilized on anitrocellulose membrane. The membrane was incubated with [¹²⁵I]-labelledC3met overnight and bound protein was visualized with a Personal FX(Bio-Rad) using intensifying screens. The recombinant proteinMID⁹⁶²⁻¹²⁰⁰ was included as a negative control. (B) UspA1⁵⁰⁻⁷⁷⁰,UspA2³⁰⁻⁵³⁹ and a series of truncated UspA2 proteins were coated onmicrotiter plates and incubated with C3met, followed by incubation withgoat anti-human C3 pAb and HRP-conjugated anti-goat pAb. The mean valuesout of three experiments are shown. The background binding wassubtracted from all samples. Error bars correspond to S.D. * P≦0.05, **P≦0.01, *** P≦0.001.

FIG. 17 illustrates that addition of recombinant UspA1⁵⁰⁻⁷⁷⁰ andUspA2³⁰⁻⁵³⁹ to serum inhibit C3b deposition and killing of M.catarrhalis via the alternative pathway. Flow cytometry profiles showC3b-deposition on RH4ΔuspA1/A2 after incubation with (A) NHS or NHSpreincubated with recombinant (rec.) UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹, or (B)NHS-Mg-EGTA or NHS-Mg-EGTA preincubated with UspA1⁵⁰⁻⁷⁷⁰ andUspA2³⁰⁻⁵³⁹. After addition of the various NHS combinations, bacteriawere analyzed as described in FIG. 13. (C) RH4ΔuspA1/A2 was incubatedwith 10% NHS or NHS-Mg-EGTA. For inhibition, the NHS-Mg-EGTA wasincubated with 100 nM UspA1⁵⁰⁻⁷⁷⁰ and/or UspA2³⁰⁻⁵³⁰ before addition ofbacteria. Bacteria were collected at the indicated time points. Thenumber of bacteria at the initiation of the experiments was defined as100%. Mean values of three separate experiments are shown and error barsindicate S.D. The time points 10, 20 and 30 min for the ΔuspA1/A2 mutantpreincubated with recombinant proteins were significantly different fromthe ΔuspA1/A2 mutant incubated with Mg-EGTA alone (P<0.05).

FIG. 18 illustrates that recombinant UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹decrease haemolysis of rabbit erythrocytes by inhibition of thealternative pathway. NHS was incubated with or without 100 nMUspA1⁵⁰⁻⁷⁷⁰ and/or UspA2³⁰⁻⁵³⁹ at 37° C. for 30 min. NHS at theindicated concentrations was thereafter added to rabbit erythrocytes.After incubation for 30 min, the suspensions were centrifuged and thesupernatants were measured by spectrophotometry. Maximum haemolysis ineach experiment was defined as 100%. Mean values of three separateexperiments are shown and error bars correspond to S.D. The resultsobtained with NHS+UspA2³⁰⁻⁵³⁹ and NHS+UspA1⁵⁰⁻⁷⁷⁰/UspA2³⁰⁻⁵³⁹ at NHSconcentrations of 2, 3 and 4% were significantly different from the NHScontrol (P<0.05).

FIG. 19 illustrates a pileup-analysis of UspA1 for eight differentstrains, to show the homology of different parts of UspA1.

FIG. 20 illustrates a pileup-analysis of UspA2 for thirteen differentstrains to show the homology of different parts of UspA2.

FIG. 21 illustrates %identity in regions identified on Forsgren sequencecomputed as the ratio between the number of exact matches and the lengthof the region alignment, where the region alignment is that part of theabove total alignment containing the Forsgren region.

MATERIALS AND METHODS

Interaction Between M. catarrhalis and Fibronectin

Bacterial Strains and Culture Conditions

The sources of the clinical M. catarrhalis strains are listed in table7. M. catarrhalis BBH18 and RH4 mutants were constructed as previouslydescribed.[23, 58] The M. catarrhalis strains were routinely cultured inbrain heart infusion (BHI) liquid broth or on BHI agar plates at 37° C.The UspA1-deficient mutants were cultured in BHI supplemented with 1.5μg/m1 chloramphenicol (Sigma, St. Louis, Mo.), and UspA2-deficientmutants were incubated with 7 μg/m1 zeocin (Invitrogen, Carlsbad,Calif.). Both chloramphenicol and zeocin were used for growth of thedouble mutants.

TABLE 7 Clinical strains of M. catarrhalis used in the present studyStrain Clinical Source Reference BBH18 Sputum [53] D1 Sputum [53] Ri49Sputum [53] C10 Sputum [10] F16 Sputum [10] Bro2 Respiratory tract [53]Z14 Pharynx [10] S6-688 Nasopharynx [23] Bc5 Nasopharynx [20] RH4 Blood[53] RH6 Blood [53] R14 Unknown [10] R4 Unknown [10] SÖ-1914 Tympaniccavity aspirate [23] Note: The strains C10, R4 did not have the uspA1gene, whereas F16, R14, Z14 lacked the uspA2 gene.[10] The remainingstrains contained both uspA1 and A2 genes (data not shown).

DNA Method

To detect the presence uspA1, A2, and A2H genes in those strains whichthis was unknown, primers and PCR conditions as described by Meier etal. was used.[50] Partial sequencing was also carried out with theUspA1²⁹⁹⁻⁴⁵² and UspA2¹⁶⁵⁻³¹⁸ 5′ and 3′ primers of the respective uspA1and uspA2 gene of RH4 and BBH18. Confirmation of the presence of theamino acid residues “DQKADIDNNINNIYELAQQQDQHSSDIKTLK” was also performedby PCR with a primer (5′-CAAAGCTGACATCCAAGCACTTG-3′) designed from the5′ end of this sequence and 3′ primers for uspA1 and A2 as described byMeier at al.[50]

Recombinant Proteins Construction and Expression

Recombinant UspA1⁵⁰⁻⁷⁷⁰ and UspA²³⁰⁻⁵³⁹ , which are devoid of theirhydrophobic C-termini, has recently been described.[58] The genomic DNAwas extracted from M. catarrhalis Bc5 using a DNeasy tissue kit (Qiagen,Hilden, Germany). In addition, recombinant proteins corresponding tomultiple regions spanning UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ were alsoconstructed by the same method. The primers used are listed in table 8.All constructs were sequenced according to standard methods. Expressionand purification of the recombinant proteins were done as describedpreviously.[59] Proteins were purified using columns containing a nickelresin (Novagen) according to the manufacturer's instructions for nativeconditions. The recombinant proteins were analyzed on SDS-PAGE asdescribed.[21]

TABLE 8 Primers used in this present study Protein 5′ primer 3′ primerUspA1⁵⁰⁻⁷⁷⁰ gcgtctgcggatccagtag ccctgaagctttagtgca gcaaggcaacctaacctaattg UspA1⁵⁰⁻⁴⁹¹ gcgtctgcggatccagtag ttgagcaagcttagcttggcaaggcaacc gtttttagcg UspA1⁵⁰⁻¹⁹⁷ gcgtctgcggatccagtagacctgtggcaagcttctt gcaaggcaacc cctgcc UspA1⁵⁰⁻³²¹ gcgtctgcggatccagtagggtgtcactaagcttacc gcaaggcaacc tgcaccaacatgaac UspA1²⁹⁹⁻⁴⁵²gatttgcaggtgcatcgga gtcttttgtaagatcaag tcctggtaatggtact cttttgatcaatUspA1⁴³³⁻⁵⁸⁰ catagctctgatatggatc catgctgagaagcttacc cacttaaaaac tagattggUspA1⁵⁵⁷⁻⁷⁰⁴ gccaaagcacaagcggatc ggtcttattggtagtaag caaataaagaccttagcttggttttg UspA1⁶⁸⁰⁻⁷⁷⁰ gttgagcaaaaggatccca ccctgaagctttagtgcatcaatcaagag taacctaattg UspA2³⁰⁻⁵³⁹ cgaatgcggatcctaaaaacattaagcttggtgtcta tgatataactttagagg atgcagttac UspA2³⁰⁻¹⁷⁷cgaatgcggatcctaaaaa ctcatgaccaaaatcaag tgatataactttagaggcttatcttcgatagactc UspA2¹⁰¹⁻²⁴⁰ gatattgcggatccggaag gatcaataagcttaccgcatgatgttgaaac ttagattgaatagttctt c UspA2¹⁰¹⁻³¹⁸ gatattgcggatccggaaggtcaatcgcttcaagctt atgatgttgaaac cttttgagcatactg UspA2¹⁶⁵⁻³¹⁸gagattgagaaggatccag gtcaatcgcttcaagctt atgctattgct cttttgagcatactgUspA2³⁰²⁻⁴⁵⁸ gctcaaaaccaagcggatc ggtgagcgtttcaagctt cccaagatctgtgcatcagcatcggc UspA2⁴⁴⁶⁻⁵³⁹ gcaagtgctgcggatcctg cattaagcttggtgtctaatcgtattgct atgcagttac

Antibodies

Rabbit anti-UspA1/A2 polyclonal antibodies (pAb) were recently describedin detail.[58] The other antibodies used were rabbit anti-humanfibronectin pAb, swine FITC-conjugated anti-rabbit pAb, swinehorseradish peroxidase (HRP) conjugated anti-rabbit pAb and finally amouse anti-human CD54 (ICAM1) monoclonal antibody (mAb). Antibodies werefrom Dakopatts (Glostrup, Denmark).

Flow Cytometry Analysis

The UspA1/A2-protein expression and the capacity of M. catarrhalis tobind fibronectin were analyzed by flow cytometry. M. catarrhalis wildtype strains and UspA1/A2-deficient mutants were grown overnight andwashed twice in phosphate buffered saline containing 3% fish gelatin(PBS-gelatin). The bacteria (10⁸) were then incubated with theanti-UspA1/A2 antiserum or 5 pg fibronectin (Sigma, St Louis, Mo.). Theywere then washed and incubated for 30 min at room temperature (RT) withFITC-conjugated anti-rabbit pAb (diluted according to the manufacturer'sinstructions) or with 1/100 dilution of rabbit anti-human fibronectinpAb (if fibronectin was first added) for 30 min at RT before incubationwith the FITC-conjugated anti-rabbit pAb. After three additional washes,the bacteria were analyzed by flow cytometry (EPICS, XL-MCL, Coulter,Hialeah, Fla.). All incubations were kept in a final volume of 100 μlPBS-gelatin and the washings were done with the same buffer.Anti-fibronectin pAb and FITC-conjugated anti-rabbit pAb were addedseparately as a negative control for each strain analyzed. Fibronectininhibition studies were carried out by pre-incubating 0.25 μmoles ofUspA fragments for 1 h with 2 μg of fibronectin before incubation withM. catarrhalis bacteria (10⁸). The residual free amount of fibronectinthat bound to M. catarrhalis was determined by flow cytometry asoutlined above.

Binding of M. catarrhalis to Immobilized Fibronectin

Glass slides were coated with 30 p1 aliquots of fibronectin (1 mg/ml)and air dried at RT. After washing once with PBS, the slides wereincubated in Petri dishes with pre-chilled bacteria at late exponentialphase (optical density (OD) at 600 nm=0.9). After 2 h at RT, glassslides were washed once with PBS followed by Gram staining.

Protein Labeling and Radio Immunoassay (RIA)

Fibronectin was ¹²⁵Iodine labeled (Amersham, Buckinghamshire, England)to a high specific activity (0.05 mol iodine per mol protein) with theChloramine T method.[21] M. catarrhalis strains BBH18 and RH4 togetherwith their corresponding mutants were grown overnight on solid mediumand were washed in PBS with 2% bovine serum albumin (BSA). Bacteria(10⁸) were incubated for 1 h at 37° C. with ¹²⁵I-labeled fibronectin(1600 kcpm/sample) in PBS containing 2% BSA. After three washings withPBS 2% BSA, ¹²⁵I-labeled fibronectin bound to bacteria was measured in agamma counter (Wallac, Espoo, Finland).

Enzyme-Linked Immunosorbent Assay (ELISA)

Microtiter plates (Nunc-Immuno Module; Roskilde, Denmark) were coatedwith 40 nM of purified recombinant UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ proteinsin 75 mM sodium carbonate, pH 9.6 at 4° C. overnight. Plates were washedfour times with washing buffer (50 mM Tris-HCl, 0.15 M NaCl, and 0.1%Tween 20, pH 7.5) and blocked for 2 h at RT with washing buffercontaining 3% fish gelatin. After four additional washings, the wellswere incubated for 1 h at RT with fibronectin (120 μg/ml) diluted inthree-fold step in 1.5% fish gelatin (in wash buffer). Thereafter, theplates were washed and incubated with rabbit anti-human fibronectin pAbfor 1 h. After additional washings, HRP-conjugated anti-rabbit pAb wasadded and incubated for 1 h at RT. Both the antihuman fibronectin andHRP-conjugated anti-rabbit pAb were diluted 1:1,000 in washing buffercontaining 1.5% fish gelatin. The wells were washed four times and theplates were developed and measured at OD₄₅₀. ELISAs with truncatedproteins spanning UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ were performed with fixeddoses of fibronectin at 80 μg/ml and 120 μg/ml, respectively.

Cell Line Adherence Inhibition Assay

Chang conjunctival cells (ATCC CCL 20.2) were cultured in RPMI 1640medium (Gibco BRL, Life Technologies, Paisley,

Scotland) supplemented with 10% fetal calf serum, 2 mM L-glutamine, and12 μg of gentamicin/ml. On the day before adherence inhibitionexperiments, cells were harvested, washed twice in gentamicin-free RPMI1640, and added to 96 well tissue culture plates (Nunc) at a finalconcentration of 10⁴ cells/well in 200 μl of gentamicin-free culturemedium. Thereafter, cells were incubated overnight at 37° C. in ahumidified atmosphere of 5% CO₂and 95% air. On the day of experiments,inhibition of M. catarrhalis adhesion was carried out by pre-incubatingincreasing concentration of recombinant UspA1/A2 truncated proteinscontaining the fibronectin binding domains (UspA1²⁹⁹⁻⁴⁵²andUspA2¹⁶⁵⁻³¹⁸) or rabbit anti-human fibronectin pAb (diluted 1:50) for 1h. Nonfibronectin binding recombinant proteins (UspA1⁴³³⁻⁵⁸⁰ andUspA2³⁰⁻¹⁷⁷) were used as controls. Chang epithelial cells are known toexpress ICAM1.[18] Hence an anti-ICAM1 antibody was used todifferentiate if the inhibitory effect of the anti-fibronectin antibodywas secondary to steric hindrance. Subsequently, M. catarrhalis RH4(10⁶) in PBS-gelatin was inoculated onto the confluent monolayers. Inall experiments, tissue culture plates were centrifuged at 3,000×g for 5min and incubated at 37° C. in 5% CO₂. After 30 min, infected monolayerswere rinsed several times with PBS-gelatin to remove non-adherentbacteria and were then treated with trypsin-EDTA (0.05% trypsin and 0.5mM EDTA) to release the Chang cells from the plastic support.Thereafter, the resulting cell/bacterium suspension was seeded indilution onto agar plates containing BHI and incubated overnight at 37°C. in 5% CO₂.

Determination of Fibronectin Expression in Chang Conjunctival EpithelialCells

Chang conjunctival epithelial cells were harvested by scraping followedby re-suspension in PBS-gelatin. Cells (1×10⁶/m1) were labeled withrabbit anti-human fibronectin pAb followed by washing and incubationwith a FITC-conjugated anti-rabbit pAb. After three additional washes,the cells were analyzed by flow cytometry as outlined above.

Interaction Between M. catarrhalis and Laminin

Bacterial Strains and Culture Conditions

The clinical M. catarrhalis strains BBH18 and RH4 and theircorresponding mutants were previously described.[58] Both strains have arelatively higher expression of UspA2 compared to UspA1.[58] The mutantsexpressed equal amount of M. catarrhalis immunoglobulin D-bindingprotein (MID) when compared to wild type strains. Bacteria wereroutinely cultured in brain heart infusion (BHI) broth or on BHI agarplates at 37° C. The UspA1-deficient, UspA2-deficient and double mutantswere cultured in BHI supplemented with antibiotics as described.[58]

Recombinant Protein Construction and Expression

Recombinant UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ , which are devoid of theirhydrophobic C-termini, were manufactured.[58] In addition, recombinantproteins corresponding to multiple regions spanning UspA1⁵⁰⁻⁷⁷⁰ andUspA2³⁰⁻⁵³⁹ were used.[78]

Antibodies

Rabbit anti-UspA1/A2 and anti-MID polyclonal antibodies (pAb) wereused.[22, 58] Rabbit anti-laminin pAb was from Sigma (St Louis, Mo.,USA). Swine horseradish peroxidase (HRP)-conjugated anti-rabbit pAb wasfrom Dakopatts (Glostrup, Denmark).

Binding of M. catarrhalis to Immobilized Laminin

Microtiter plates (Nunc-Immuno Module; Roskilde, Denmark) were coatedwith Engelbreth-Holm-Swarm mouse sarcoma laminin (Sigma, Saint Louis,USA) or bovine serum albumin (BSA) (30 μg/ml) in Tris-HCL, pH 9.0 at 4°C. overnight. The plates were washed with phosphate buffered saline and0.05% Tween 20, pH 7.2 (PBS-Tween) and subsequently blocked with 2% BSAin PBS+0.1% Tween 20, pH 7.2. M. catarrhalis RH4 and BBH18 (10⁸) in 100μl were then added followed by incubation for 1 h. Unbound bacteria wereremoved by washing 3 times with PBS-Tween. Residual bound bacteria weredetected by means of an anti-MID pAb, followed by detection withHRP-conjugated anti-rabbit pAb. The plates were developed and measuredat 0D4₅₀ according to a standard protoccol.

Enzyme-Linked Immunosorbent Assay (ELISA)

Microtiter plates (Nunc-Immuno Module) were coated with 40 nM ofpurified recombinant UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ proteins in 75 mMsodium carbonate, pH 9.6 at 4° C. Plates were washed four times withwashing buffer (50 mM Tris-HCl, 0.15 M NaCl, and 0.1% Tween 20, pH 7.5)and blocked at RT with washing buffer containing 3% fish gelatin. Afteradditional washings, the wells were incubated for 1 h at RT with lamininat different dilutions as indicated in 1.5% fish gelatin (in washbuffer). Thereafter, the plates were washed and incubated with rabbitanti-laminin pAb. After additional washings, HRP-conjugated anti-rabbitpAb was added and incubated at RT. Both the anti-laminin andHRP-conjugated anti-rabbit pAb were diluted 1:1,000 in washing buffercontaining 1.5% fish gelatin. The wells were washed and the plates weredeveloped and measured at OD₄₅₀. Uncoated wells incubated with identicaldilutions of laminin were used as background controls. ELISAs withtruncated proteins spanning UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ were performedwith fixed doses of laminin (20 μg/ml).

Interaction Between M. catarrhalis and C3 and C3met

Bacterial Strains and Culture Conditions

The clinical M. catarrhalis isolates and related subspecies haverecently been described in detail.[21, 53] Type strains were from theCulture Collection, University of Gothenburg (CCUG; Department ofClinical Bacteriology, Sahlgrenska Hospital, Gothenburg, Sweden), or theAmerican Type Culture Collection (ATCC; Manassas, Va.); Neisseriagonorrheae CCUG 15821, Streptococcus pyogenes CCUG 25570 and 25571,Streptococcus agalactiae CCUG 4208, Streptococcus pneumoniae ATCC 49619,Legionella pneumophila ATCC 33152, Pseudomonas aeruginosa ATCC 10145,Staphylococcus aureus ATCC 29213, and finally Staphylococcus aureus ATCC25923. The remaining strains in Table 9 were clinical isolates fromMedical Microbiology, Department of Laboratory Medicine, MalmöUniversity Hospital, Lund University, Sweden.

TABLE 9 M. catarrhalis is a unique C3/C3met binding bacterium. Relatedmoraxella subspecies and other common human pathogens do not bindC3/C3met (mfi < 2.0). After incubation with EDTA-treated NHS or C3met,bacteria were analysed by flow cytometry using a rabbit anti-C3d pAb anda FITC-conjugated goat anti-rabbit pAb. Species NHS-EDTA (mfi) C3met(mfi) Moraxella catarrhalis RH4 8.7 22.1 M. osloensis <2.0 <2.0 M. bovis<2.0 <2.0 M. caniculi <2.0 <2.0 M. nonliquefacie <2.0 <2.0 N. pharyngis<2.0 <2.0 N. sicca <2.0 <2.0 N. flava <2.0 <2.0 N. subflava <2.0 <2.0Oligella ureolytica (n = 2) <2.0 <2.0 Haemophilus influenzae (n = 7)<2.0 <2.0 Streptococcus pneumoniae (n = 11) <2.0 <2.0 Legionellapneumophila (n = 2) <2.0 <2.0 Pseudomonas aeruginosa (n = 2) <2.0 <2.0Listeria monocytogenes <2.0 <2.0 Yersinia entercolitica <2.0 <2.0Staphylococcus aureus (n = 3) <2.0 <2.0 Streptococcus pyogenes (n = 2)<2.0 <2.0 Streptococcus agalactia <2.0 <2.0 Enterococcus faecalis <2.0<2.0 Helicobacter pylori <2.0 <2.0 Escherichia coli (n = 2) <2.0 <2.0 M.ovis <2.0 <2.0 M. caviae <2.0 <2.0 Neisseria gonorrheae <2.0 <2.0 N.meningtidis <2.0 <2.0 N. mucosa <2.0 <2.0

The different non-moraxella species were grown on appropriate standardculture media. M. catarrhalis strains were routinely cultured in brainheart infusion (BHI) liquid broth or on BHI agar plates at 37° C. M.catarrhalis BBH18 and RH4 mutants were manufactured as previouslydescribed. [22, 23, 58] The MID-deficient mutants were grown in BHIcontaining 50 μg/ml kanamycin. The UspA1-deficient mutants were culturedin BHI supplemented with 1.5 μg/ml chloramphenicol (Sigma, St. Louis,Mo.), and UspA2-deficient mutants were incubated with 7 μg/ml zeocin(Invitrogen, Carlsbad, Calif.). Both chloramphenicol and zeocin wereused for growth of the UspA1/A2 double mutants.

Antibodies

Rabbits were immunized intramuscularly with 200 μg recombinantfull-length UspA1 emulsified in complete Freunds adjuvant (Difco, BectonDickinson, Heidelberg, Germany), and boosted on days 18 and 36 with thesame dose of protein in incomplete Freunds adjuvant.[22] Blood was drawn3 weeks later. To increase the specificity, the anti-UspA1 antiserum wasaffinity-purified with Sepharose-conjugated recombinant UspA1⁵⁰⁻⁷⁷⁰.[58]The antiserum bound equally to UspA1 and UspA2 and was thus designatedanti-UspA1/A2 pAb. The rabbit anti-human C3d pAb and the FITC-conjugatedswine anti-rabbit pAb were purchased from Dakopatts (Glostrup, Denmark),and the goat anti-human C3 were from Advanced Research Technologies (SanDiego, Calif.). The horseradish peroxidase (HRP)-conjugated donkeyanti-goat pAb was obtained from Serotec (Oxford, UK).

Proteins and Iodine Labelling

The manufacture of recombinant UspA1⁵⁰³⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹, which aredevoid of their hydrophobic C-termini, has recently been described.[23]The truncated UspA1 and UspA2 proteins were manufactured as described indetail by Tan et al.[78] C3b was purchased from Advanced ResearchTechnologies. C3(H₂O) was obtained by freezing and thawing of purifiedC3. The C3b-like molecule (C3met) was made by incubation of purified C3with 100 mM methylamine (pH 8.0) for 2 h at 37° C., and subsequentdialysis against 100 mM Tris-HCl (pH 7.5), 150 mM NaCl. For bindingstudies, C3met was labelled with 0.05 mol ¹²⁵I (Amersham,Buckinghamshire, England) per mol protein, using the Chloramine Tmethod.[25]

Flow Cytometry Analysis

Binding of C3 to M. catarrhalis and other species was analyzed by flowcytometry. Bacteria were grown on solid medium overnight and washedtwice in PBS containing 2% BSA (Sigma) (PBS-BSA). Thereafter, bacteria(10⁸ colony forming units; cfu) were incubated with C3met, C3b, C3(H₂O), or 10% NHS with or without 10 mM EDTA or 4 mM MgCl₂ and 10 mMEGTA (Mg-EGTA) in PBS-BSA for 30 min at 37° C. After washings, thebacteria were incubated with anti-human C3d pAb for 30 min on ice,followed by washings and incubation for another 30 min on ice withFITC-conjugated goat anti-rabbit pAb. After three additional washes,bacteria were analyzed by flow cytometry (EPICS, XL-MCL, Coulter,Hialeah, Fla.). All incubations were kept in a final volume of 100 μlPBS-BSA and the washings were done with the same buffer. The anti-humanC3d pAb and FITC-conjugated anti-rabbit pAb were added separately as anegative control for each strain analyzed. In the inhibition studies,serum was preincubated with 100 nM of the recombinant UspA1⁵⁰⁻⁷⁷⁰ andUspA2³⁰⁻⁵³⁹ proteins for 30 min at 37° C. To analyze the characteristicsof the M. catarrhalis and C3 interaction, increasing concentrations ofNaCl (0-1.0 M) was added to bacteria and C3met. To analyze UspA1/A2expression, bacteria (10⁸ cfu) were incubated with the anti-UspA1/A2 pAband washed as described above. A FITC-conjugated goat anti-rabbit pAbdiluted according to the manufacturers instructions was used fordetection. To assure that EDTA did not disrupt the outer membraneproteins UspA1 and UspA2, M. catarrhalis was incubated with or withoutEDTA followed by detection of UspA1/A2 expression. EDTA, at theconcentrations used in the NHS-EDTA experiments, did not change thedensity of UspA1/A2.

Serum and Serum Bactericidal Assay

Normal human serum (NHS) was obtained from five healthy volunteers. Theblood was allowed to clot for 30 min at room temperature and thereafterincubated on ice for 60 min. After centrifugation, sera were pooled,aliquoted and stored at −70° C. To inactivate both the classical andalternative pathways, 10 mM EDTA was added. In contrast, Mg-EGTA wasincluded to inactivate the classical pathway. Human serum deficient inthe C4BP was prepared by passing fresh serum through a HiTrap column(Amersham Biosciences) coupled with mAb 104, a mouse mAb directedagainst CCP1 of the α-chain of C4BP.[41] The flow through was collectedand the depleted serum was stored in aliquots at −70° C. Serum depletedof C1q was obtained via the first step of C1q purification [79] usingBiorex 70 ion exchange chromatography (Bio-Rad, Hercules, Calif.). Theresulting sera displayed normal haemolytic activity. The factor D andproperdin deficient serum was kindly provided by Dr. Anders Sjöholm(Department of Medical Microbiology, Lund University, Lund, Sweden). M.catarrhalis strains were diluted in 2.5 mM Veronal buffer, pH 7.3containing 0.1% (wt/vol) gelatin, 1 mM MgCl₂, 0.15 mM CaCl₂, and 2.5%dextrose (DGVB⁺⁺). Bacteria (10³ cfu) were incubated together with 10%NHS and EDTA or Mg-EGTA in a final volume of 100 μl. The bacteria/NHSwas incubated at 37° C. and at various time points, 10 μl aliquots wereremoved and spread onto BHI agar plates. In inhibition studies, 10%serum was incubated with 100 nM of the recombinant UspA1⁵⁰⁻⁷⁷⁰ andUspA2³⁰⁻⁵³⁹ proteins for 30 min at 37° C. before bacteria were added.

Dot Blot Assays

Purified recombinant UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ diluted in three-foldsteps (1.9-150 nM) in 100 μl of 0.1 M Tris-HCl, pH 9.0 were applied tonitrocellulose membranes (Schleicher & Schüll, Dassel, Germany) using adot blot device. After saturation, the membranes were incubated for 2 hwith PBS-Tween containing 5% milk powder at room temperature and washedfour times with PBS-Tween. Thereafter, 5 kcpm [125I]-labelled C3met inPBS-Tween with 2% milk powder was added overnight at 4° C. The boundprotein was visualized with a Personal FX (Bio-Rad) using intensifyingscreens.

Surface Plasmon Resonance (Biacore)

The interaction between UspA1⁵⁰⁻⁷⁷⁰ or UspA2³⁰⁻⁵³⁹ and C3 was furtheranalysed using surface plasmon resonance (Biacore 2000; Biacore,Uppsala, Sweden) as recently described for the UspA1/2-C4BPinteraction.[58] The K_(D) (the equilibrium dissociation constant) wascalculated from a binding curve showing response at equilibrium plottedagainst the concentration using steady state affinity model supplied byBiaevaluation software (Biacore).

Enzyme-Linked Immunosorbent Assay (ELISA)

Microtiter plates (Nunc-Immuno Module; Roskilde, Denmark) were coatedwith triplets of purified recombinant UspA1⁵⁰⁻⁷⁷⁰, UspA2³⁰⁻⁵³⁹, or thetruncated UspA1 and UspA2 fragments (40 nM in 75 mM sodium carbonate, pH9.6) at 4° C. overnight. Plates were washed four times with washingbuffer (PBS with 0.1% Tween 20, pH 7.2) and blocked for 2 hrs at roomtemperature with washing buffer supplied with 1.5% ovalbumin (blockingbuffer). After washings, the wells were incubated overnight at 4° C.with 0.25 μg C3met in blocking buffer. Thereafter, the plates werewashed and incubated with goat anti-human C3 in blocking buffer for 1 hat RT. After additional washings, HRP-conjugated donkey anti-goat pAbswas added for another 1 h at RT. The wells were washed four times andthe plates were developed and measured at OD₄₅₀.

Haemolytic Assay

Rabbit erythrocytes were washed three times with ice-cold 2.5 mM Veronalbuffer, pH 7.3 containing 0.1% (wt/vol) gelatin, 7 mM MgCl₂, 10 mM EGTA,and 2.5% dextrose (Mg⁺⁺EGTA), and resuspended at a concentration of0.5×10⁹ cells/ml. Erythrocytes were incubated with variousconcentrations (0 to 4%) of serum diluted in Mg⁺⁺EGTA. After 1 h at 37°C., erythrocytes were centrifuged and the amount of lysed erythrocyteswas determined by spectro-photometric measurement of released hemoglobinat 405 nm. For inhibition with UspA1 and UspA2, 10% serum waspreincubated with 100 nM of recombinant UspA1⁵⁰⁻⁷⁷⁰ and/or UspA2³⁰⁻⁵³⁹proteins for 30 min at 37° C., and thereafter added to the erythrocytesat 0 to 4%.

Isolation of Polymorphonuclear Leukocytes and Phagocytosis

Human polymorphonuclear leukocytes (PMN) were isolated from fresh bloodof healthy volunteers using macrodex (Pharmalink AB, Upplands Väsby,Sweden). The PMN were centrifuged for 10 min at 300 g, washed in PBS andresuspended in RPMI 1640 medium (Life Technologies, Paisley, Scotland).The bacterial suspension (0.5×10⁸) was opsonized with 3% of either NHSor NHS-EDTA, or 20 μg of purified C3met for 15 min at 37° C. Afterwashes, bacteria were mixed with PMN (1×10⁷ cells/ml) at a bacteria/PMNratio of 10:1 followed by incubation at 37° C. with end-over-endrotation. Surviving bacteria after 0, 30, 60, and 120 min of incubationwas determined by viable counts. The number of engulfed NHS-treatedbacteria was compared with bacteria phagocytosed in the absence of NHS.S. aureus opsonized with NHS was used as positive control.

Examples and Results

Interaction Between M. catarrhalis and Fibronectin M. catarrhalis Devoidof UspA1 and A2 does not Bind Soluble or Immobilized Fibronectin.

We selected a random series of M. catarrhalis clinical strains (n=13)(table 7) and tested them for fibronectin binding in relation to theirUspA1/A2 expression by flow cytometry analysis. High UspA1/A2 expressionas determined by high mean fluorescence intensity (MFI) was correlatedto UspA1/A2 expression (Pearson correlation coefficient 0.77, P<0.05)(FIG. 1A). However, to discriminate between UspA1 and A2 expression wasnot possible with our anti-UspA1/A2 pAb. Moreover, the presence ofUspA2H protein contributing to the binding was unlikely as the uspA2Hgene was not found in the strains used in this study (data not shown).

Two M. catarrhalis isolates (BBH18 and RH4) and their specific mutantslacking UspA1, UspA2 or both proteins were also analyzed by flowcytometry. M. catarrhalis BBH18 strongly bound fibronectin with a meanfluorescence intensity (MFI) of 96.1 (FIG. 1F). In contrast, BBH18ΔuspA1showed a decreased fibronectin binding with an MFI of 68.6 (FIG. 1G).Fibronectin binding to BBH18ΔuspA2 and the double mutant BBH18ΔuspA1/A2revealed an MFI of only 10.7 and 11.5, respectively (FIG. 1H, 1I).Similar results were obtained with UspA1/A2 mutants of the clinicalstrain M. catarrhalis RH4. Taken together, these results suggest thatUspA1 and A2 bound fibronectin and that the ability of the bacteria tobind fibronectin strongly depended on UspA1/A2 expression.

To further analyze the interaction between fibronectin and M.catarrhalis, ¹²⁵I-labeled fibronectin was incubated with two clinical M.catarrhalis isolates (BBH18 and RH4) and their respective mutants. Thewild type M. catarrhalis RH4 strongly bound ¹²⁵I-fibronectin while thecorresponding ΔuspA1 mutant showed 80% binding of the wild type. Incontrast, the ΔuspA2 and double mutant bound ¹²⁵I-fibronectin at 14% and12%, respectively, which was just above the background levels (5.0 to10%) (FIG. 2). Similar results were obtained with M. catarrhalis BBH18and the corresponding UspA1/A2 mutants. Thus, our results suggest thatboth UspA1 and A2 are required for the maximal binding of solublefibronectin by M. catarrhalis.

To investigate the bacterial attachment to immobilized fibronectin, M.catarrhalis RH4 and its corresponding ΔuspA1/A2 mutants were appliedonto fibronectin coated glass slides. After 2 h of incubation, slideswere washed, and subsequently Gram stained. M. catarrhalis wild type andthe ΔuspA1 mutant were found to strongly adhere to the fibronectincoated glass slides (FIGS. 3A and 3B). In contrast, M. catarrhalisΔuspA2 and ΔuspA1/A2 double mutants weakly adhered to the fibronectincoated glass slide with only a few bacteria left after washing (FIGS. 3Cand 3D, respectively). Experiments with another M. catarrhalis clinicalisolate (BBH18) and its derived mutants showed a similar patternindicating that UspA2 was of major importance for M. catarrhalis bindingto immobilized fibronectin.

The Fibronectin Binding Domains Include Amino Acid Residues LocatedBetween 299 and 452 of UspA1 and Between 165 and 318 of UspA2

To further analyze the interactions of UspA1 and A2 with fibronectin,truncated UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ were recombinantly produced in E.coli, coated on microtiter plates and incubated with increasingconcentrations of fibronectin. Bound fibronectin was detected with ananti-human fibronectin pAb followed by incubation with a horseradishperoxidase conjugated anti-rabbit pAb. Both recombinant UspA1⁵⁰⁻⁷⁷⁰ andUspA2³⁰⁻⁵³⁹ bound soluble fibronectin and the interactions weredose-dependent (FIG. 4).

To define the fibronectin-binding domain of UspA1, recombinant proteinsspanning the entire molecule of UspA1⁵⁰⁻⁷⁷⁰ were manufactured.Fibronectin was incubated with the immobilized UspA1 proteins fragmentsand the interactions were quantified by ELISA. UspA1⁵⁰⁻⁴⁹¹ boundfibronectin almost as efficiently as UspA1⁵⁰⁻⁷⁷⁰ suggesting that thebinding domain was within this part of the protein. Among the othertruncated fragments, UspA1²⁹⁹⁻⁴⁵² efficiently bound fibronectin (FIG.5A). In parallel, the interactions between fibronectin and severalrecombinant UspA2 fragments including amino acids UspA2³⁰⁻⁵³⁹wereanalyzed. The two fragments UspA2¹⁰¹⁻³¹⁸ and UspA2¹⁶⁵⁻³¹⁸ strongly boundfibronectin (FIG. 5B). Our findings provide significant evidence thatthe binding domains include residues found within UspA1²⁹⁹⁻⁴⁵² andUspA2¹⁶⁵⁻³¹⁸. A sequence comparison between these two binding fragmentsrevealed that the 31 amino acid residues“DQKADIDNNINNIYELAQQQDQHSSDIKTLK” were identical for UspA1 and A2 (FIG.6). Moreover, this repeat sequence was also found in the uspA1 and A2gene of M. catarrhalis BBH18 and RH4 (data not shown).

UspA1⁵⁰⁻⁴⁹¹ and UspA1²⁹⁹⁻⁴⁵² Fragments Competitively inhibit M.catarrhalis Fibronectin Binding

To further validate our findings on the UspA1/A2 fibronectin bindingdomains, recombinant truncated UspA1 proteins were tested for theircapacity to block fibronectin binding to M. catarrhalis. Fibronectin (2pg) was pre-incubated with 0.25 μmoles of recombinant UspA1 fragmentsand subsequently incubated with M. catarrhalis. Finally, M. catarrhalisUspA-dependent fibronectin binding was measured by flow cytometry.Pre-incubation with UspA1⁵⁰¹⁻⁴⁹¹ and UspA1²⁹⁹⁻⁴⁵² resulted in decreasedfibronectin binding with a 95% reduction for UspA1⁵⁰⁻⁴⁹¹ and a 63%reduction for UspA 1299-452 (FIG. 7). When fibronectin was pre-incubatedwith the truncated UspA2¹⁰¹⁻³¹⁸ an inhibition of 50% was obtained.

Thus, the fibronectin binding domains of UspA1 and A2 block theinteractions between fibronectin and M. catarrhalis.

UspA1²⁹⁹⁻⁴⁵² and UspA2¹⁶⁵⁻³¹⁸ Inhibit M. catarrhalis Adherence to ChangEpithelial Cells

Epithelial cells are known to express fibronectin and many bacteriaattach to epithelial cells via cell-associated fibronectin.[46, 54, 69,77] Previous studies have shown that M. catarrhalis adhere to epithelialcells.[43, 49] We analyzed Chan conjunctival cells, which havefrequently been used in adhesion experiments with respiratory pathogens.Chang cells strongly expressed fibronectin as revealed by flow cytometryanalysis (FIG. 8A).

To analyze whether the UspA-dependent fibronectin binding was importantfor bacterial adhesion, Chang epithelial cells were pre-incubated withanti-human fibronectin pAb, or the recombinant proteins UspA1²⁹⁹⁻⁴⁵² andUspA2¹⁶⁵⁻³¹⁸. Thereafter, M. catarrhalis RH4 was added and bacterialadhesion analyzed. The relative adherence (measured by the number ofcolony forming units) after pre-incubation with 0.4 μmoles per 200 μl ofUspA1²⁹⁹⁻⁴⁵², UspA2¹⁶⁵⁻³¹⁸, or an anti-human fibronectin pAb were 36%,35% and 32%, respectively. Higher concentrations of recombinant peptidesdid not result in further inhibition. In contrast, the non-fibronectinbinding fragments UspA1⁴³³⁻⁵⁸⁰ and UspA2³⁰⁻¹⁷⁷ did not inhibit theinteractions between M. catarrhalis and the Chang epithelial cells (FIG.8B). Thus, fibronectin on Chang epithelial cells may function as areceptor for M. catarrhalis and the amino acid residues 299-452 of UspA1and 165-318 of UspA2 contain the ligand responsible for theinteractions.

Interaction Between M. catarrhalis and Laminin M. catarrhalis bindsLaminin through UspA1 and A2

Two clinical M. catarrhalis isolates (BBH18 and RH4) and their specificmutants lacking UspA1, UspA2 or both proteins were analyzed by awhole-cell ELISA. M. catarrhalis RH4 strongly bound to immobilizedlaminin. (FIG. 9A). In contrast, M. catarrhalis RH4 uspA1 mutant(RH4ΔuspA1) showed a laminin binding of 89.9% of the wild type. M.catarrhalis RH4 uspA2 mutant (RH4ΔuspA2) and the double mutantRH4ΔuspA1/A2 15.2% and 18.1% binding capacity of the wild type,respectively. This was not significantly different from the residualadhesion to BSA coated plates. Similar results were obtained withUspA1/A2 mutants originating from the clinical strain M. catarrhalisBBH18. In these two strains (BBH18 and RH4), UspA2 is the predominantprotein expressed as compared to UspA1, explaining the minimaldifference in binding between the wild type and RH4ΔuspA1. Takentogether, these results show that UspA1 and A2 bound laminin.

To further analyze the binding between UspA1/A2 and laminin, truncatedUspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ were produced in E. coli. Recombinantproteins were coated on microtiter plates and incubated with increasingconcentrations of laminin. Bound laminin was detected with a rabbitanti-laminin pAb followed by incubation with an HRP-conjugatedanti-rabbit pAb. Both recombinant UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ stronglybound soluble laminin and the binding was dose-dependent and saturable(FIG. 9B).

To define the laminin binding domains, recombinant UspA1 and A2 spanningthe entire molecules were manufactured. Laminin was incubated withimmobilized truncated UspA1 and A2 fragments and followed byquantification by ELISA. UspA1⁵⁰⁻⁴⁹¹ bound to laminin almost asefficiently as UspA1⁵⁰⁻⁷⁷⁰ suggesting that the binding domain was withinthis part of the protein. However, among the other truncated fragmentsspanning this region, no other fragment appeared to bind laminin. TheN-terminal part, UspA2³⁰⁻³⁵¹, was able to retain 44.7% binding capacityas compared to the full length protein. The shorter protein UspA2³⁰⁻¹⁷⁷showed a 43.7% binding capacity. (FIG. 10B). These results show that thebinding domains include residues found within the N-terminals of bothUspA1 and UspA2.

Interaction Between M. catarrhalis and C3 and C3met M. catarrhalis OuterMembrane Proteins UspA1 and UspA2 Inhibit Both the Classical and theAlternative Pathway of the Complement Cascade

UspA2 surface expression is crucial for M. catarrhalis survival innormal human serum (NHS) [1, 58], i.e., moraxella UspA2 deficientmutants are rapidly killed when exposed to NHS. We have recently shownthat both UspA1 and A2 bind C4BP and thus might inhibit the classicalpathway of complement activation [58]. To further shed light on M.catarrhalis interactions with the complement system, survival ofUspA1/A2 double mutants was studied in serum treated with either EGTAwith addition of MgCl₂ (Mg-EGTA) or EDTA. Mg-EGTA inhibits the classicaland lectin pathways and thus allows separate analysis of the alternativepathway. In contrast, EDTA inhibits all complement pathways by absorbingdivalent cations (Mg²⁺ and Ca²⁺). The M. catarrhalis RH4 wild typesurvived after 30 min of incubation, whereas RH4ΔuspA1/A2 double mutantwas killed by intact NHS after 10 min (FIG. 12). When the classicalpathway was inhibited (NHS+Mg-EGTA), the RH4ΔuspA1/A2 mutant survivedfor a significantly longer period of time as compared to NHS without anychelators, but not as long as the wild type bacterium. Furthermore, whenboth the classical and alternative pathways were blocked with EDTA, M.catarrhalis RH4ΔuspA1/A2 survived. A similar pattern was obtained withthe M. catarrhalis BBH18 isolate and the corresponding BBH18 ΔuspA1/A2mutants (not shown). In parallel, experiments with C1q and factorD/properdin deficient sera demonstrated that both the classical and thealternative pathways were inhibited by M. catarrhalis (not shown). Thus,M. catarrhalis, a pathogen that frequently colonizes the humanrespiratory tract, does not only counteract the classical pathway butalso the alternative pathway of the complement system by the outermembrane proteins UspA1 and A2.

M. catarrhalis Absorbs C3 from EDTA-Inactivated Serum

C3b covalently binds to the surface of a microbe and hence induces thealternative pathway (FIG. 11B). To analyze whether M. catarrhalis caninteract with C3, our RH4 wild type strain was incubated with NHS or NHStreated with EDTA. Binding or deposition (via covalent link) of C3/C3bat the bacterial surface of M. catarrhalis RH4 was detected by flowcytometry analysis with a polyclonal antibody (pAb) directed against C3drecognizing both C3 and C3b. Incubation of bacteria with NHS containingintact complement led to deposition of C3 (FIG. 13). Interestingly, whenthe complement cascade was inactivated in the presence of EDTA, the M.catarrhalis RH4 still bound C3 (FIG. 13A). Streptococcus pneumoniae thatwas included for comparison did not absorb C3 from the EDTA-treatedserum (FIG. 13B). In contrast to pneumococci, M. catarrhalis thus boundC3 irrespectively of complement activation. The internal thioester of C3is spontaneously hydrolysed in fluid phase to C3(H20). Thus, intact C3or C3(H₂O) was the most likely forms of C3 interacting with M.catarrhalis. Since M. catarrhalis also binds C4BP [58], we wanted toexclude that C4BP was involved in the C3 binding and for that purpose weused C4BP depleted serum. M. catarrhalis absorbed C3 from the C4BPdepleted serum to the same extent as to NHS (not shown).

Binding of C3met to M. catarrhalis is Dose-Dependent and Non-Covalent

Our experiments implied that C3 bound to the surface of M. catarrhalisirrespectively of complement activation. Therefore, we analyzed whetherconverted C3, which is non-functional, could bind to the bacteria.Native C3 was purified from human serum and treated with methylamine,which converts C3 to a C3met molecule equivalent to C3b without thecapacity to covalently bind to microbes (FIG. 11C). Flow cytometryanalysis revealed that the M. catarrhalis RH4 wild type strainefficiently bound C3met in a dose-dependent and saturable manner (FIGS.14A and B). This interaction was not mediated by the C3a part of the C3molecule since C3b and C3 (H₂O) also bound M. catarrhalis (not shown).The binding between M. catarrhalis RH4 and C3met was based to a largeextent on ionic interactions as increasing concentrations of NaClinhibited the interaction (FIG. 14C). Similar results were obtained withthe M. catarrhalis BBH18 wild type strain (not shown).

To determine whether the binding of C3 is a general feature of all M.catarrhalis strains, we selected a random series of clinical isolates(n=13) and analyzed their capacity to bind C3met. All M. catarrhalisstrains bound C3met as revealed by a flow cytometry analysis with ananti-C3d pAb. The mfi values varied from 4 to 39. However, S. pneumoniaeand E. coli that were included for comparison did not bind C3met.

M. catarrhalis is a Unique C3 and C3met binding Bacterium

To extend our analysis of bacterial C3 absorption from

NHS, related moraxella subspecies (n=13) as well as common humanpathogens (n=13) were incubated in the presence of NHS-EDTA.Interestingly, among all the bacterial species tested, M. catarrhaliswas the only bacterium binding C3 in complement-inactivated serum (Table9). All related moraxella strains as well as the other human pathogenswere also analyzed for binding of C3met. In parallel with the C3binding, M. catarrhalis was the only species that bound C3met. Takentogether, M. catarrhalis has a unique feature to strongly bind C3 andC3met in a non-covalent manner.

M. catarrhalis binds C3met via the Outer Membrane Proteins UspA1 andUspA2

To determine the M. catarrhalis protein responsible for the C3 binding,we tested a series of bacterial mutants devoid of the outer membraneproteins MID, UspA1 and/or UspA2 [22, 58]. Interestingly, the binding ofC3met was significantly correlated with Usp expression (FIG. 15). M.catarrhalis RH4Δmid bound C3met to the same degree as the wild typecounterpart (FIG. 15A-B). The RH4ΔuspA1 mutant showed only a slightlydecreased binding, whereas the RH4ΔuspA2 was a weaker binder as comparedto the wild type counterpart (FIG. 15C-D). In parallel, C3met binding tothe double RH4ΔuspA1/A2 mutant was completely abolished (FIG. 15E).Furthermore, when the same experiments were performed using NHS-EDTA,the same pattern was seen (FIG. 15F-J). When normal human serum wasused, all mutants showed similar amount of C3 on their surface since itwas a mixture of covalent deposition and binding of C3 (FIG. 15K-O).Similar results were obtained with the M. catarrhalis BBH18 isolate andthe corresponding BBH18 mutants.

To further analyze the interaction between C3 and UspA1/A2, UspA1⁵⁰⁻⁷⁷⁰and UspA2³⁰⁻⁵³⁹ were produced in E. coli and purified. The recombinantproteins were dot blotted onto a nitrocellulose membrane followed byincubation with iodine-labelled C3met. Recombinant MID⁹⁶²⁻¹²⁰⁰, which isderived from the M. catarrhalis outer membrane protein MID [59], wasincluded as a negative control. A weak binding to UspA1^(5D-770) wasdetected, whereas [¹²⁵I]-C3met strongly bound to UspA2³⁰⁻⁵³⁹ (FIG. 16A).These findings were further strengthened using surface plasmon resonance(i.e., Biacore). UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ were immobilized on thesurface of a CM5 chip using amino coupling and C3met was injected untilsaturation was reached. The KD for the interaction between C3met andUspA2³⁰⁻⁵³⁹ or UspA1⁵⁰⁻⁷⁷⁰ was 3 and 14 μM, respectively. In conclusion,we found that UspA2 was the major C3met-binding protein of M.catarrhalis, whereas UspA1 contributed to the binding to a lower degree.

A C3 Binding Domain is Located Between Amino Acid Residues 200 and 458of UspA2.

To define the C3 binding domain of UspA2, recombinant proteins spanningthe entire UspA2³⁰⁻⁵³⁹ molecule were manufactured. C3met was incubatedwith the immobilized full length UspA1⁵⁰⁻⁷⁷⁰, UspA2³⁰⁻⁵³⁹ and a seriesof truncated UspA2 proteins. Thereafter, the interactions werequantified by ELISA. In agreement with the dot blot experiments (FIG.16A), UspA1⁵⁰⁻⁷⁷⁰ bound C3met to a much lower extent compared toUspA2³⁰⁻⁵³⁹ in the ELISA (FIG. 16B). Among the truncated proteinfragments, UspA2^(165-318,) UspA2²⁰⁰⁻⁵³⁹ and UspA2³⁰²⁻⁴⁵⁸ efficientlybound C3met, suggesting that a binding domain was within the amino acidresidues 200 and 458.

Recombinant UspA1/A2 Neutralizes C3 Activity

In order to in detail examine the role of UspA1/A2-dependent inhibitionof the alternative pathway, a series of flow cytometry experiments wasperformed with bacteria incubated with 10% NHS or serum that had beenpreincubated with 100 nM recombinant UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹.Interestingly, a significantly decreased C3 deposition/binding at thesurface of M. catarrhalis RH4ΔuspA1/A2 was observed when NHS waspretreated with UspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ (FIG. 17A). When theclassical pathway was shut down with Mg-EGTA, similar results wereobtained (FIG. 17B). Thus, the recombinant proteins UspA1⁵⁰⁻⁷⁷⁰ andUspA2³⁰⁻⁵³⁹ absorbed C3 from NHS and inhibited deposition/binding of C3.

To determine whether absorption of C3 by recombinant UspA1⁵⁰⁻⁷⁷⁰ andUspA2³⁰⁻⁵³⁹ increased bacterial survival, the double mutant M.catarrhalis RH4ΔuspA1/A2 was incubated with serum supplemented withUspA1⁵⁰⁻⁷⁷⁰and UspA2³⁰⁻⁵³⁹ followed by determination of the number ofsurviving bacteria. Mg-EGTA was included in the reactions in order toinhibit the classical pathway. Interestingly, addition of recombinantUspA1⁵⁰⁻⁷⁷⁰ and UspA2³⁰⁻⁵³⁹ to NHS prevented killing of the UspA1/A2deficient M. catarrhalis (FIG. 17C). UspA2³⁰⁻⁵³⁹ was most efficient ininhibiting bacterial killing as compared to UspA1⁵⁰⁻⁷⁷⁰ . When bothrecombinant proteins were supplemented together, no additionalinhibition of the alternative pathway was detected. Ten % NHS correspondto approximately 600 nM C3. To investigate whether more UspA1 moleculescould neutralize the C3 activity, UspA1⁵⁰⁻⁷⁷⁰ and/or UspA2³⁰⁻⁵³⁹ up to600 nM was added. However, higher concentrations of the recombinantproteins did not further increase the inhibition (not shown).

We also included an alternative pathway haemolytic assay consisting ofrabbit erythrocytes and NHS in order to establish the role of UspA1 andA2 as inhibitors of the alternative pathway. NHS was preincubated withrecombinant UspA1⁵⁰⁻⁷⁷⁰ UspA2³⁰⁻⁵³⁹, or both proteins together followedby addition to the erythrocytes. After 1 h incubation, the amount oferythrocyte lysis was determined. Interestingly, a significantlydecreased haemolysis was observed when NHS was preincubated withUspA1⁵⁰⁻⁷⁷⁰ or UspA2³⁰⁻⁵³⁹ as compared to untreated NHS (FIG. 18). Inparallel with the increased survival of bacteria in the presence ofUspA2³⁰⁻⁵³⁹ or UspA1⁵⁰⁻⁷⁷⁰ (FIG. 17C), preincubation with UspA2³⁰⁻⁵³⁹alone resulted in a more efficient inhibition of the alternative pathwayas compared to when NHS was preincubated with UspA1⁵⁰⁻⁷⁷⁰. Inconclusion, recombinant UspA1⁵⁰⁻⁷⁷⁰ or UspA2³⁰⁻⁵³⁹ interfered with theactivity of the alternative pathway due to their ability to capture C3.

In addition of being a key molecule in the complement cascade, depositedC3b and iC3b (inactivated C3b) target microbes for removal in theprocess of opsonophagocytosis. To investigate whether 03 or C3met thatwas non-covalently bound at the surface of M. catarrhalis could stillfunction as an opsonin, a series of phagocytosis experiments wasperformed. M. catarrhalis was preincubated with C3met, NHS or NHStreated with EDTA followed by addition of polymorphonuclear leukocytes.Interestingly, M. catarrhalis was not engulfed in the presence of C3met,whereas NHS strongly promoted phagocytosis (data not shown). However,when NHS was pretreated with EDTA, M. catarrhalis was not phagocytosedby polymorphonuclear leukocytes. Thus, C3/C3met was inactive at the M.catarrhalis cell surface and did not function as an opsonin.

Discussion

Interaction between M. catarrhalis and Fibronectin

UspA1²⁹⁹⁻⁴⁵² and UspA2¹⁶⁵⁻³¹⁸ from the clinical M. catarrhalis strainBc5 were the shortest fragments that still bound fibronectin.Interestingly, longer fragments encompassing the amino acid sequencefound within UspA1²⁹⁹⁻⁴⁵² and UspA2¹⁶⁵⁻³¹⁸ displayed a more efficientbinding to fibronectin (FIGS. 5A and B). This may mean that these tworegions represent partial binding domains or that the binding site ishighly dependent on a specific molecular structure. UspA1²⁹⁹⁻⁴⁵² andUspA2¹⁶⁵⁻³¹⁸ share a sequence of 31 identical amino acid residuesincluding the 23 residues “NNINNIYELAQQQDQHSSDIKTL” (NNINNIY sequence).This sequence contains the epitope for the protective monoclonalantibody (mAb) 17C7 for which there is universal reactivity.[2, 50, 30]In a mouse model, passive immunization with mAb 17C7 provided protectionand improved pulmonary clearance of M. catarrhalis.[30] It is hence mostinteresting that UspA1/A2 fibronectin binding domains contain theseresidues and argues for the importance of this region in thepathogenesis of M. catarrhalis respiratory tract infection.

The fibronectin binding M. catarrhalis BBH18 and RH4 used in ourexperiments also carry the 31 amino acid residues in their UspA1/A2protein. Most M. catarrhalis have a part of this sequence (i.e., theNNINNIY sequence). However, strains like the 035E which has the NNINNIYsequence in their UspA2 gene do not express a fibronectin binding UspA2protein.[49] A likely explanation would be that the variations in theflanking regions might affect the interaction with fibronectin. Also,the conserved NNINNIY sequence itself can have minor single amino acidbase changes.[28] It is thus likely that fibronectin binding woulddepend not just on UspA1/A2 expression, but also on the individualmakeup of each UspA protein. Interestingly, an almost identical aminoacid sequence can be found in the hybrid UspA2H protein with adhesiveproperties (M. catarrhalis TTA37 and O46E).[43] This give support to ourfindings that the 31 amino acid sequence is important in adhesion.

In our last set of experiments, we tested whether the adherence of M.catarrhalis to Chang conjunctival cells could be inhibited by thefibronectin binding fragments (UspA1²⁹⁹⁻⁴⁵² and UspA2¹⁶⁵⁻³¹⁸) (FIG. 8B).Preincubation with UspA1²⁹⁹⁻⁴⁵², UspA2¹⁶⁵⁻³¹⁸ or an anti-fibronectin pAbresulted in decreased binding to Chang epithelial cells. These resultsconfirm the importance of these binding domains in the interactions ofUspA1/A2 with Chang epithelial cells and further suggest thatfibronectin is an important receptor for UspA. In addition, it is knownthat FnBP facilitate the adherence of bacteria to undifferentiated andinjured airways.[54, 69] Fibronectin expression by lung fibroblasts isalso increased by cigarette smoke extract.[87] The role of M.catarrhalis UspA1/A2 binding to ECM fibronectin or epithelialcell-associated fibronectin is thus of great importance in patients withCOPD and may explain the common occurrence of M. catarrhalis infectionin this group of patients.[40]

In conclusion, we have shown that UspA1/A2 of M. catarrhalis BBH18, RH4and Bc5 are crucial FnBP. Both recombinant UspA1 and A2 derived from Bc5bind fibronectin with a binding domain sharing identical amino acidresidues including the conserved NNINNIY sequence. Furthermore, aninteraction of M. catarrhalis UspA1/A2 with epithelial cells is viacell-associated fibronectin. The definition of these fibronectin bindingdomains is therefore an important step forward in the development of avaccine against M. catarrhalis.

Interaction Between M. catarrhalis and Laminin

M. catarrhalis is a common cause of infectious exacerbations in patientswith COPD. The success of this species in patients with COPD is probablyrelated in part to its large repertoire of adhesins. In addition, thereare pathological changes such as loss of epithelial integrity withexposure of basement membrane where the laminin layer itself isthickened in smokers.[4] Some pathogens have been shown to be able tobind to laminin and thus may contribute to their ability to adhere tosuch damaged and denuded mucosal surfaces. These include pathogens knownto cause significant disease in the airways such as S. aureus and P.aeruginosa amongst others.[7, 63]

We recently showed that both UspA1 and A2 bind fibronectin.[78] Thefibronectin binding domains were located within UspA1²⁹⁹⁻⁴⁵² andUspA2¹⁶⁵⁻³¹⁸. In this study, the N-terminal halves UspA1⁵⁰⁻⁴⁹¹ andUspA2³⁰⁻³⁵¹ (containing the fibronectin domains) also bound laminin.However, the smallest fragments that bound fibronectin, UspA1²⁹⁹⁻⁴⁵² andUspA2¹⁶⁵⁻³¹⁸ did not bind laminin to any appreciable extent. In fact,fragments smaller than the N-terminal half of UspA1 (UspA1⁵⁰⁻⁴⁹¹) lossesall its laminin binding ability whereas with UspA2, only UspA2³⁰⁻¹⁷⁰bound laminin albeit at a lower level then the whole recombinant protein(UspA2³⁰⁻⁵³⁹). These findings suggest that perhaps different parts ofthe molecules might have different functional roles.

Comparing the smallest laminin binding regions of UspA1 and A2, we findthat there is, however, little similarity by way of amino acid homologybetween UspA2³⁰⁻¹⁷⁰ and UspA1⁵⁰-491 (data not shown). This is notsurprising as it is a known fact that both proteins have a‘lollipop’-shaped globular head structure despite having only 22%identity in both N-terminal halves.[2, 32] We postulate that a tertiarystructure is likely responsible for the interactions with laminin in thehead region in vivo. The localization of the binding domains at theN-terminal end would be logical as this would be most exposed and incontact with the human basement membrane in vivo.

Bacterial factors mediating adherence to tissue and extracellular matrix(ECM) components are grouped together in a single family named“microbial surface components recognizing adhesive matrix molecules”(MSCRAMMS). Since UspA1/A2 bind both fibronectin and laminin, theseproteins can be designated MSCRAMMS. Our results suggest that UspA1 andA2 are multifunctional adhesins with different domains interacting withdifferent ligands in the respiratory tract. Similar broad-spectrumbinding profiles have been reported for other bacterial proteins such asYadA of Yersinia enterocolitica for which UspA1 and A2 bear a structuralrelationship. [45, 70] YadA too binds both fibronectin and laminin. [32]

In summary we have shown that UspA1/A2 are crucial to M. catarrhalisinteraction with the basement membrane glycoprotein laminin and thiswill play an important role in the pathogenesis of infections inpatients with COPD. [74]

Interaction Between M. catarrhalis and C3 and C3met

Complement resistance is one of the most important bacterial virulencefactors.[66] The majority (89%) of M. catarrhalis isolates from patientswith lower respiratory tract infections are resistant tocomplement-mediated killing.[34] M. catarrhalis UspA1 and A2 are crucialfor bacterial survival in human serum in vivo [1, 15], and we have shownthat these two outer membrane proteins bind to the complement fluidphase regulator of the classical pathway, C4BP.[58] In the presentstudy, we demonstrate that M. catarrhalis can inhibit the alternativepathway by non-covalently binding of C3 (FIGS. 17 and 18). The bindingof C3 most likely also inhibits the classical pathway. This could,however, not be analysed in detail since M. catarrhalis also binds C4BP.Interestingly, the M. catarrhalis-dependent C3-binding is unique asseveral related moraxella subspecies as well as common human pathogenicbacteria do not bind C3 (Table 9). The interactions with C3 andmethylamine-treated C3 are mediated mainly by UspA2, whereas UspA1 has aminor role (FIGS. 15 and 16). The C3-binding region of UspA2 waslocalized between the amino acid residues 200 to 458. This regioncontains a stretch of 140 amino acid residues that is 93% identical to aregion in UspA1.[2] However, despite this sequence similarity, UspA1binds C3 to a much lower extent. This might be due to a specificdifference in conformation between the proteins. The discrepancy in theC3 binding of UspA1 and UspA2 stands in contrast to the UspA1/A2interaction with C4BP.[58]

M. catarrhalis is equally resistant to both the classical andalternative pathways (FIG. 12B). The bacterium binds C4BP that inhibitsthe classical pathway [58] and in this paper we demonstrate aninteraction with the alternative pathway through binding of C3. Todetermine which of these mechanisms that is of most importance for theM. catarrhalis serum resistance in various in vivo situations isdifficult. For example, the importance of the classical pathway willstrongly depend on history of infections with M. catarrhalis and abilityto generate complement-activating antibodies. However, every mechanismproviding protection from the complement is certainly beneficial for apathogen. Since C3 is a key molecule in the complement system, thebinding of C3 most likely results in regulation of all three activationpathways and may contribute the most to serum resistance.

The importance of the complement system as a primary defence mechanismis mirrored by the fact that microbes have developed various strategiesto interfere with and/or neutralize components of the complementsystem.[42, 35, 88] In addition to M. catarrhalis, S. pyogenes,Bordetella pertussis, E. coli K1, Candida albicans, and N. gonorrhoeaeexpress specific surface molecules that bind C4BP and as a consequenceprotect the bacteria against the classical complement pathway.[8, 9, 52,58, 64, 65, 80] In addition to inhibition of the classical pathway,several bacteria (e.g., C. albicans, N. meningitides, S. pyogenes, andS. pneumoniae; for reviews see [68, 89] bind factor H and factor H-likemolecule and hence are partially protected against the alternativecomplement pathway.

UspA1 and A2 absorb C3 from serum and hereby most likely inhibit thecomplement activation. Similarly, the Pneumococcal Surface Protein A(PspA) appears to inhibit the alternative pathway both in vitro and invivo. PspA is an important virulence factor for S. pneumoniae.PspA-deficient pneumococcal strains are readily cleared from the blood,whereas the PspA-expressing strains survive.[82] Furthermore, in amurine model of bacteremia, PspA-deficient pneumococci have asignificantly reduced virulence compared with pneumococci that expressPspA.[11] It has been demonstrated that more C3b is deposited onPspA-negative pneumococci than on PspA-positive.[67, 82] Thus,expression of PspA reduces the complement-mediated clearance andphagocytosis of S. pneumoniae by limiting opsonization by C3b.[12, 67]PspA-deficient pneumococci that are not virulent in normal mice becomevirulent in C3-deficient and factor B-deficient mice.[82]

To our knowledge, there are only two examples of bacterial proteins thatnon-covalently bind C3 and thereby interfere with complement function.The first one is the extracellular fibrinogen-binding protein (Efb) ofStaphylococcus aureus, which was found to bind 03b.[44] Efb inhibitsboth the classical and alternative pathways independently of thethioester conformation, i.e., the binding to C3b is non-covalent. Thesecond example is the pneumococcal choline-binding protein (CbpA), whichhas been shown to bind methylamine-treated C3, suggesting a non-covalentinteraction that is not dependent on complement activation.[16] CbpA isa component of the pneumococcal cell wall, but may only bind C3 when theCbpA is secreted. In order to test this hypothesis, which is not firmlyestablished in the literature, we analyzed eleven different pneumococcalisolates for C3 binding (methylamine-treated C3 or NHS-EDTA) by flowcytometry (FIG. 12B and Table 9). No bound C3 could be detected on thesurface of S. pneumoniae. When lysates of S. pneumoniae and culturesupernatants were analyzed on Western blots using methylamine-treated C3followed by an anti-human C3 pAb, we confirmed the results by Cheng andcollaborators [16] (not shown). In the light of Efb and CbpA, which bothare C3-binding proteins secreted by two Gram-positive bacteria, theGram-negative M. catarrhalis is a unique species with membrane anchoredproteins that bind C3 and inhibit the alternative pathway at the surfaceof the bacterium.

The yeast Candida albicans has been shown to bind C3b, iC3b and C3d.However, C3b is bound at a considerably lower affinity than iC3b andC3d.[29] We found a large difference between C3 binding to M.catarrhalis and C. albicans (not shown); despite that candida boundC3met (56% positive cells), the mean fluorescence intensity (mfi) wasonly <2.0 as compared to mfi 36.9 for M. catarrhalis. Furthermore, nodetectable binding was seen when C. albicans was incubated withEDTA-treated serum. Two C3d-binding proteins have been isolated from C.albicans and the most characterized protein is a 60 kDa mannoproteinthat initially was recognized by an antibody directed against humancomplement receptor 2 (CD21).[13] However, M. catarrhalis UspA1 and A2were not recognized by a polyclonal antibody directed against CD21 (notshown). In parallel with staphylococci and pneumococci [52, 64], asecreted C3d-binding protein from C. albicans also exists.[72] Finally,a C. albicans iC3b receptor has been isolated and is structurallysimilar to human CR3 (CD11b).[3] The mechanisms by which these receptorsmay participate in pathogenesis are not fully known.

The above examples of C3 binding pathogens are notably different from M.catarrhalis in that these species often are blood stream isolates. M.catarrhalis is mucosal pathogen with rare instances of bacteremicinfections. Hence, the binding and inactivating C3 most likely occur atthe mucosal surface. This is supported by the fact that there is strongongoing complement activation and consequent inflammation in diseasestate such as acute otitis media.[57] The complement proteins arebelieved to be transported to the mucosal surface due to exudation ofplasma.[26, 62] In middle-ear effusions (MEEs) from children forexample, strongly elevated concentrations of C3 products can also befound.[51] In addition, complement factors in MEEs fluid have been shownto be important in the bactericidal activity against other mucosalagents such as non-typable H. influenzae.[75]M. catarrhalis is a stricthuman pathogen. It does not cause diseases such as otitis media orpneumonia in animals. A mouse pulmonary clearance model and an otitismedia model with chinchilla has been used at several occasions. However,neither otitis media nor pneumonia develops and bacteria are rapidlycleared.[19, 83] It is thus difficult to test the biologicalsignificance of bacterial C3 binding in vivo. Since UspA1 and A2 aremultifunctional proteins [1, 15, 31, 43, 58, 78], it would be impossibleto relate any differences in the clearance of M. catarrhalis to C3binding. In particular the fact that UspA1 is an important adhesin of M.catarrhalis and binds both CEACAM1 and fibronectin [31, 78] would mostlikely affect the clearance. Nevertheless, due to the strong complementactivation in disease states such as otitis media, moraxella-dependentbinding of C3 may represent an important way of combating the mucosaldefense.

The fact that M. catarrhalis hampers the human immune system in severalways might explain why M. catarrhalis is such a common inhabitant of therespiratory tract [73]. In conclusion, M. catarrhalis has developedsophisticated ways of combating both the humoral and innate immunesystems. The present data show that M. catarrhalis has a uniqueC3-binding capacity at the bacterial cell surface that cannot be foundin other bacterial species.

REFERENCES

-   1. Aebi C, Lafontaine E R, Cope L D, et al. Phenotypic effect of    isogenic uspA1 and uspA2 mutations on Moraxella catarrhalis O35E.    Infect Immun 1998; 66:3113-9.-   2. Aebi C, Maciver I, Latimer J L, et al. A protective epitope of    Moraxella catarrhalis is encoded by two different genes. Infect    Immun 1997; 65:4367-77.-   3. Alaei, S., C. Larcher, C. Ebenbichler, W. M. Prodinger, J.    Janatova, and M. P. Dierich. 1993. Isolation and biochemical    characterization of the iC3b receptor of Candida albicans. Infect.    Immun. 61:1395-1399.-   4. Amin K, Ekberg-Jansson A, Lofdahl C G and Venge P.

Relationship between inflammatory cells and structural changes in thelungs of asymptomatic and never smokers: a biopsy study. Thorax 2003;58:135-42.

-   5. Attia A S, Lafontaine E R, Latimer J L, Aebi C, Syrogiannopoulos    G A and Hansen E J. The UspA2 protein of Moraxella catarrhalis is    directly involved in the expression of serum resistance. Infect    Immun 2005; 73:2400-10.-   6. Bartos L C, Murphy T F. Comparison of the outer membrane proteins    of 50 strains of Branhamella catarrhalis. J Infect Dis 1988;    158:761-5.-   7. de Bentzmann S, Tristan A, Etienne J, Brousse N, Vandenesch F and    Lina G. Staphylococcus aureus isolates associated with necrotizing    pneumonia bind to basement membrane type I and IV collagens and    laminin. J Infect Dis 2004; 190:1506-15.-   8. Berggård, K., E. Johnsson, F. R. Mooi, and G. Lindahl. 1997.    Bordetella pertussis binds the human complement regulator C4BP: role    of filamentous hemagglutinin. Infect. Immun. 65:3638-3643.-   9. Blom, A. M., A. Rytkonen, P. Vasquez, G. Lindahl, B. Dahlbäck,    and A. B. Jonsson. 2001. A novel interaction between type IV pili of    Neisseria gonorrhoeae and the human complement regulator C4B-binding    protein. J. Immunol. 166:6764-6770.-   10. Bootsma H J, van der Heide H G, van de Pas S, Schouls L M and    Mooi F R. Analysis of Moraxella catarrhalis by DNA typing: evidence    for a distinct subpopulation associated with virulence traits. J    Infect Dis 2000; 181:1376-87.-   11. Briles, D. E., J. Yother, L. S. McDaniel. 1988. Role of    pneumococcal surface protein A in the virulence of Streptococcus    pneumoniae. Rev. Infect. Dis. 10:(Suppl. 2):S372.-   12. Briles, D. E., S. K. Hollingshead, E. Swiatlo, A.    Brooks-Walter, A. Szalai, A. Virolainen, L. S. McDaniel, K. A.    Benton, P. White, K. Prellner, A. Hermansson, P. C. Aerts, H. Van    Dijk, and M. J. Crain. 1997. PspA and PspC: their potential for use    as pneumococcal vaccines. Microb. Drug Resist. 3:401-408.-   13. Calderone, R. A., L. Linehan, E. Wadsworth, and A. L.    Sandberg. 1988. Identification of C3d receptors on Candida albicans.    Infect. Immun. 56:252-258.-   14. Catlin B W. Branhamella catarrhalis: an organism gaining respect    as a pathogen. Clin Microbiol Rev 1990; 3:293-320.-   15. Chen D, Barniak V, VanDerMeid K R and McMichael J C. The levels    and bactericidal capacity of antibodies directed against the UspA1    and UspA2 outer membrane proteins of Moraxella (Branhamella)    catarrhalis in adults and children. Infect Immun 1999; 67:1310-6.-   16. Cheng, Q., D. Finkel, and M. K. Hostetter. 2000. Novel    purification scheme and functions for a C3-binding protein from    Streptococcus pneumoniae. Biochem. 39:5450-5457.-   17. Cope L D, Lafontaine E R, Slaughter C A, et al. Characterization    of the Moraxella catarrhalis uspA1 and uspA2 genes and their encoded    products. J Bacterial 1999; 181:4026-34.-   18. De Saint Jean M, Baudouin C, Di Nolfo M, et al. Comparison of    morphological and functional characteristics of primary-cultured    human conjunctival epithelium and of Wong-Kilbourne derivative of    Chang conjunctival cell line. Exp Eye Res 2004; 78:257-74.-   19. Fulghum, R. S., and H. G. Marrow. 1996. Experimental otitis    media with Moraxella (Branhamella) catarrhalis. Ann. Otol. Rhinol.    Laryngol. 105:234-241.-   20. Forsgren, A., and A. Grubb. 1979. Many bacterial species bind    human IgD. J. Immunol 122:1468-1472.-   21. Forsgren A, Brant M, Mollenkvist A, et al. Isolation and    characterization of a novel IgD-binding protein from Moraxella    catarrhalis. J Immunol 2001; 167:2112-20.-   22. Forsgren A, Brant M, Karamehmedovic M and Riesbeck K.

The immunoglobulin D-binding protein MID from Moraxella catarrhalis isalso an adhesin. Infect Immun 2003; 71:3302-9.

-   23. Forsgren A, Brant M and Riesbeck K. Immunization with the    truncated adhesion moraxella catarrhalis immunoglobulin D-binding    protein (MID764-913) is protective against M. catarrhalis in a mouse    model of pulmonary clearance. J Infect Dis 2004; 190:352-5.-   24. Gjörloff-Wingren, A., R. Hadzic, A. Forsgren, and K.    Riesbeck. 2002. A novel IgD-binding bacterial protein from Moraxella    catarrhalis induces human B lymphocyte activation and isotype    switching in the presence of Th2 cytokines. J. Immunol.    168:5582-5588.-   25. Greenwood F C, Hunter W M and Glover J S. The Preparation of    I-131-Labelled Human Growth Hormone of High Specific Radioactivity.    Biochem J 1963; 89:114-23.-   26. Greiff, D., I. Erjefält, C. Svensson, P. Wollmer, U. Alkner, M.    Andersson, and C. G. Persson. 1993. Plasma exudation and solute    absorbtion across the airway mucosa. Clin. Physiol. 13: 219-233.-   27. Hanski E, Caparon M. Protein F, a fibronectin-binding protein,    is an adhesin of the group A streptococcus Streptococcus pyogenes.    Proc Natl Acad Sci USA 1992; 89:6172-6.-   28. Hays J P, van der Schee C, Loogman A, et al. Total genome    polymorphism and low frequency of intra-genomic variation in the    uspA1 and uspA2 genes of Moraxella catarrhalis in otitis prone and    non-prone children up to 2 years of age. Consequences for vaccine    design? Vaccine 2003; 21:1118-24.-   29. Heidenreich, F., and M. P. Dierich. 1985. Candida albicans and    Candida stellatoidea, in contrast to other Candida species, bind    iC3b and C3d but not C3b. Infect. Immun. 50:598-600.-   30. Helminen M E, Maciver I, Latimer J L, et al. A large,    antigenically conserved protein on the surface of Moraxella    catarrhalis is a target for protective antibodies. J Infect Dis    1994; 170:867-72.-   31. Hill D J, Virji M. A novel cell-binding mechanism of Moraxella    catarrhalis ubiquitous surface protein UspA: specific targeting of    the N-domain of carcinoembryonic antigen-related cell adhesion    molecules by UspA1. Mol Microbiol 2003; 48:117-29.-   32. Hoiczyk E, Roggenkamp A, Reichenbecher M, Lupas A and    Heesemann J. Structure and sequence analysis of Yersinia YadA and    Moraxella UspAs reveal a novel class of adhesins. EMBO J 2000;    19:5989-99.-   33. Holm M M, Vanlerberg S L, Foley I M, Sledjeski D D and    Lafontaine E R. The Moraxella catarrhalis porin-like outer membrane    protein CD is an adhesin for human lung cells. Infect Immun 2004;    72:1906-13.-   34. Hol, C., C. M. Verduin, E. E. Van Dijke, J. Verhoef, A.

Fleer, H. van Dijk. 1995. Complement resistance is a virulence factor ofBranhamella (Moraxella) catarrhalis. FEMS Immunol. Med. Microbiol.11:207-211.

-   35. Hornef, M. W., M. J. Wick, M. Rhen, and S. Normark. 2002.    Bacterial strategies for overcoming host innate and adaptive immune    responses. Nat. Immunol. 3:1033-1040.-   36. Hostetter, M. K., M. L. Thomas, F. S. Rosen, and B. F.    Tack. 1982. Binding of C3b proceeds by a transesterification    reaction at the thiolester site. Nature 298:72-75.-   37. Hostetter, M. K., R. A. Krueger, and D. J. Schmeling. 1984. The    biochemistry of opsonization: central role of the reactive    thiolester of the third component of complement. J. Infect. Dis.    150:653-661.-   38. Joh D, Wann E R, Kreikemeyer B, Speziale P and Hook M. Role of    fibronectinbinding MSCRAMMs in bacterial adherence and entry into    mammalian cells. Matrix Biol 1999; 18:211-23.-   39. Joh H J, House-Pompeo K, Patti J M, Gurusiddappa S and Hook M.    Fibronectin receptors from gram-positive bacteria: comparison of    active sites. Biochemistry 1994; 33:6086-92.-   40. Karalus R, Campagnari A. Moraxella catarrhalis: a review of an    important human mucosal pathogen. Microbes Infect 2000; 2:547-59.-   41. Kask, L., L. A. Trouw, B. Dahlback, and A. M. Blom. 2004. The    C4b-binding protein-protein S complex inhibits the phagocytosis of    apoptotic cells. J. Biol. Chem. 279:23869-23873.-   42. Lachmann, P. J. 2002. Microbial subversion of the immune    response. Proc. Natl. Acad. Sci. U.S.A 99:8461-8462.-   43. Lafontaine E R, Cope L D, Aebi C, Latimer J L, McCracken G H,    Jr. and Hansen E J. The UspA1 protein and a second type of UspA2    protein mediate adherence of Moraxella catarrhalis to human    epithelial cells in vitro. J Bacterial 2000; 182:1364-73.-   44. Lee, L. Y. L., M. Hook, D. Haviland, R. A. Wetsel, E. O.    Yonter, P. Syribeys, J. Vernachio, and E. L. Brown. 2004. Inhibition    of complement activation by secreted Staphylococcus aureus    protein. J. Infect. Dis. 190:571-579-   45. Mack D, Heesemann J and Laufs R. Characterization of different    oligomeric species of the Yersinia enterocolitica outer membrane    protein YadA. Med Microbial Immunol (Berl) 1994; 183:217-27.-   46. Maheshwari R K, Kedar V P, Bhartiya D, Coon H C and Kang Y H.    Interferon enhances fibronectin expression in various cell types. J    Biol Regul Homeost Agents 1990; 4:117-24.-   47. McGavin M J, Gurusiddappa S, Lindgren P E, Lindberg M, Raucci G    and Hook M. Fibronectin receptors from Streptococcus dysgalactiae    and Staphylococcus aureus. Involvement of conserved residues in    ligand binding. J Biol Chem 1993; 268:23946-53.-   48. McMichael J C. Vaccines for Moraxella catarrhalis. Vaccine 2000;    19 Suppl 1:S101-7.-   49. McMichael J C, Fiske M J, Fredenburg R A, et al. Isolation and    characterization of two proteins from Moraxella catarrhalis that    bear a common epitope. Infect Immun 1998; 66:4374-81.-   50. Meier P S, Troller R, Grivea I N, Syrogiannopoulos G A and    Aebi C. The outer membrane proteins UspA1 and UspA2 of Moraxella    catarrhalis are highly conserved in nasopharyngeal isolates from    young children. Vaccine 2002; 20:1754-60.-   51. Meri, S., T. Lehtinen, and T. Palva. 1984. Complement in chronic    secretory otitis media. C3 breakdown and C3 splitting activity.    Arch. Otolaryngol. 110:774-778.-   52. Meri, T., A. M. Blom, A. Hartmann, D. Lenk, S. Meri, P. F.    Zipfel. 2004. The hyphal and yeast forms of Candida albicans bind    the complement regulator C4b-binding protein. Infect. Immun.    72:6633-6641.-   53. Mollenkvist A, Nordstrom T, Hallden C, Christensen J J, Forsgren    A and Riesbeck K. The Moraxella catarrhalis immunoglobulin D-binding    protein MID has conserved sequences and is regulated by a mechanism    corresponding to phase variation. J Bacteriol 2003; 185:2285-95.-   54. Mongodin E, Bajolet O, Cutrona J, et al. Fibronectin-binding    proteins of Staphylococcus aureus are involved in adherence to human    airway epithelium. Infect Immun 2002; 70:620-30.-   55. Murphy T F. Branhamella catarrhalis: epidemiology, surface    antigenic structure, and immune response. Microbiol Rev 1996;    60:267-79.-   56. Murphy T F, Brauer A L, Aebi C and Sethi S. Identification of    surface antigens of Moraxella catarrhalis as targets of human serum    antibody responses in chronic obstructive pulmonary disease. Infect    Immun 2005; 73:3471-8.-   57. Närkiö-Mäkelä, M., J. Jero, and S. Meri 1999. Complement    activation and expression of membrane regulators in the middle ear    mucosa in otitis media with effusion. Clin. Exp. Immunol.    116:401-409.-   58. Nordstrom T, Blom A M, Forsgren A and Riesbeck K. The emerging    pathogen Moraxella catarrhalis Interacts with complement inhibitor    C4b binding protein through ubiquitous surface proteins A1 and A2. J    Immunol 2004; 173:4598-606.-   59. Nordstrom T, Forsgren A and Riesbeck K. The immunoglobulin    D-binding part of the outer membrane protein MID from Moraxella    catarrhalis comprises 238 amino acids and a tetrameric structure. J    Biol Chem 2002; 277:34692-9.-   60. Nordstrom T, Blom A M, Tan T T, Forsgren A and Riesbeck K. Ionic    binding of C3 to the human pathogen Moraxella catarrhalis is a    unique mechanism for combating innate immunity. J Immunol 2005;    MS#05-2213, in press.-   61. Pearson M M, Lafontaine E R, Wagner N J, St Geme J W, 3rd and    Hansen E J. A hag mutant of Moraxella catarrhalis strain O35E is    deficient in hemagglutination, autoagglutination, and immunoglobulin    D-binding activities. Infect Immun 2002; 70:4523-33.-   62. Persson, C. G., I. Erjefält, U. Alkner, C. Baumgarten, L.    Greiff, B. Gustafsson, A. Luts, U. Pipkorn, F. Sundler, C. Svensson    et al. 1991. Plasma exudation as a first line respiratory mucosal    defence. Clin. Exp. Allergy 21: 17-24.-   63. Plotkowski M C, Tournier J M and Puchelle E. Pseudomonas    aeruginosa strains possess specific adhesins for laminin. Infect    Immun 1996; 64:600-5.-   64. Prasadarao, N. V., A. M. Blom, B. O. Villoutreix, and L. C.    Linsangan. 2002. A novel interaction of outer membrane protein A    with C4b binding protein mediates serum resistance of Escherichia    coli K1. J. Immunol. 169:6352-6360.-   65. Ram, S., M. Cullinane, A. M. Blom, S. Gulati, D. P.    McQuillen, B. G. Monks, C. O'Connell, R. Boden, C. Elkins, M. K.    Pangburn, B. Dahlback, and P. A. Rice. 2001. Binding of C4b-binding    protein to porin: a molecular mechanism of serum resistance of    Neisseria gonorrhoeae. J. Exp. Med. 193:281-296.-   66. Rautemaa, R., and S. Meri. 1999. Complement-resistance    mechanisms of bacteria. Microbes Infect. 1:785-94.-   67. Ren, B., A. J. Szalai, S. K. Hollingshead, D. E. Briles. 2004.    Effects of PspA and antibodies to PspA on activation and deposition    of complement on the pneumococcal surface. Infect. Immun.    72:114-122.-   68. Rodríguez de Córdoba, S., J. Esparza-Gordillo, E. Goicoechea de    Jorge, M. Lopez-Trascasa, and P. Sánchez-Corral. 2004. The human    complement factor H: functional roles, genetic variations and    disease associations. Mol. Immunol. 41:355-367.-   69. Roger P, Puchelle E, Bajolet-Laudinat O, et al. Fibronectin and    alpha5betal integrin mediate binding of Pseudomonas aeruginosa to    repairing airway epithelium. Eur Respir J 1999; 13:1301-9.-   70. Roggenkamp A, Ackermann N, Jacobi C A, Truelzsch K, Hoffmann H    and Heesemann J. Molecular analysis of transport and oligomerization    of the Yersinia enterocolitica adhesin YadA. J Bacteriol 2003;    185:3735-44.-   71. Rozalska B, Wadstrom T. Protective opsonic activity of    antibodies against fibronectin-binding proteins (FnBPs) of    Staphylococcus aureus. Scand J Immunol 1993; 37:575-80.-   72. Saxena, A., and R. Calderone. 1990. Purification and    characterization of the extracellular C3d-binding protein of Candida    albicans. Infect. Immun. 58:309-314.-   73. Schwarz-Linek U, Werner J M, Pickford A R, et al. Pathogenic    bacteria attach to human fibronectin through a tandem beta-zipper.    Nature 2003; 423:177-81.-   74. Sethi, S., and T. F. Murphy. 2001. Bacterial infection in    chronic obstructive pulmonary disease in 2000: a state-of-the-art    review. Clin. Microbiol. Rev. 14:336-363.-   75. Shimizu, T., T. Harada, Y. Majima, and Y. Sakakura. 1988.    Bactericidal activity of middle ear effusion on a single isolate of    non-typable Haemophilus influenzae. Int. J. Pediatr.    Otorhinolaryngol. 16:211-217.-   76. Tack, B. F., R. A. Harrison, J. Janatova, M. L. Thomas,    and J. W. Prahl. 1980. Evidence for presence of an internal    thiolester bond in third component of human complement. Proc. Natl.    Acad. Sci. U.S.A. 77: 5764-5768.-   77. Talay S R, Valentin-Weigand P, Jerlstrom PG , Timmis K N and    Chhatwal G S. Fibronectin-binding protein of Streptococcus pyogenes:    sequence of the binding domain involved in adherence of streptococci    to epithelial cells. Infect Immun 1992; 60:3837-44.-   78. Tan, T. T., T. Nordstrom, A. Forsgren, and K. Riesbeck. 2005.    The Respiratory Pathogen Moraxella catarrhalis Adheres to Epithelial    Cells by Interacting with Fibronectin through Ubiquitous Surface    Proteins A1 and A2. J. Infect. Dis., MS#33893, in press.-   79. Tenner, A. J., P. H. Lesavre, and N. R. Cooper. 1981.    Purification and radiolabeling of human C1q. J. Immunol.    127:648-653.-   80. Thern, A., L. Stenberg, B. Dahlbäck, and G. Lindahl. 1995.    Ig-binding surface proteins of Streptococcus pyogenes also bind    human C4b-binding protein (C4BP), a regulatory component of the    complement system. J. Immunol. 154:375-386.-   81. Timpe J M, Holm M M, Vanlerberg S L, Basrur V and Lafontaine    E R. Identification of a Moraxella catarrhalis outer membrane    protein exhibiting both adhesin and lipolytic activities. Infect    Immun 2003; 71:4341-50.-   82. Tu, A. H., R. L. Fulgham, M. A. McCrory, D. E. Briles, A. J.    Szalai. 1999. Pneumococcal surface protein A inhibits complement    activation by Streptococcus pneumoniae. Infect. Immun. 67:4720.-   83. Unhanand, M., I. Maciver, O, Ramilo, O, Arencibia-Mireles, J. C.    Argyle, G. H. McCracken Jr, and E. J. Hansen. 1992. Pulmonary    clearance of Moraxella catarrhalis in an animal model. J. Infect.    Dis. 165:644-650.-   84. Verduin C M, Hol C, Fleer A, van Dijk H and van Belkum A.    Moraxella catarrhalis: from emerging to established pathogen. Clin    Microbiol Rev 2002; 15:125-44.-   85. Walport, M. J. 2001. Complement. First of two parts. N. Engl. J.    Med. 344:1058-1066.-   86. Walport, M. J. 2001. Complement. Second of two parts. N.    Engl. J. Med. 344:1140-1144.-   87. Wang H, Liu X, Umino T, et al. Effect of cigarette smoke on    fibroblast-mediated gel contraction is dependent on cell density. Am    J Physiol Lung Cell Mol Physiol 2003; 284:L205-13.-   88. Wurzner, R. 1999. Evasion of pathogens by avoiding recognition    or eradication by complement, in part via molecular mimicry. Mol.    Immunol. 36:249-260.-   89. Zipfel, P. F., C. Skerka, J. Hellwage, S. T. Jokiranta, S.    Meri, V. Brade, P. Kraiczy, M. Noris, and G. Remuzzi. 2001. Factor H    family proteins: on complement, microbes and human diseases.    Biochem. Soc. Trans. 30:971-978.

1. A peptide consisting of Sequence ID No. 2, or a fragment, homologue,derivative, degenerate, a hydroxylation, sulphonation or glycosylationproduct, or other secondary processing product thereof that retainsfibronectin binding properties.
 2. A peptide consisting of Sequence IDNo. 3, or a fragment, homologue, derivative, degenerate, ahydroxylation, sulphonation or glycosylation product, or other secondaryprocessing product thereof that retains fibronectin binding properties.3. A method for the treatment or prophylaxis of an infection in anindividual, comprising administering a pharmaceutically effective amountof a composition comprising a substance according to claim
 1. 4. Amethod according to claim 3, wherein the infection is caused byMoraxella catarrhalis.
 5. A method according to claim 3, wherein theinfection is otitis media, sinusitis, or a lower respiratory tractinfection.
 6. A ligand comprising a fibronectin binding domain, saidligand consisting of an amino acid sequence selected from the groupconsisting of Sequence ID No. 2 and Sequence ID No. 3, or the ligand isa fragment, homologue, derivative, degenerate, a hydroxylation,sulphonation or glycosylation product, or other secondary processingproduct of the peptide of Sequence ID No. 2 or Sequence ID No. 3,wherein the ligand retains fibronectin binding properties.
 7. A fusionprotein comprising one or more ligands according to claim
 6. 8. Apharmaceutical composition comprising one or more ligands according toclaim 6 or a fusion protein comprising one or more ligands according toclaim 6, and one or more pharmaceutically acceptable adjuvants,vehicles, excipients, binders, carriers, or preservatives.
 9. A vaccinecomprising according to claim 8 one or more ligands according to claim 6or a fusion protein comprising one or more ligands according to claim 6,and one or more pharmaceutically acceptable adjuvants, vehicles,excipients, binders, carriers, or preservatives.
 10. A method oftreating or preventing an infection in an individual comprisingadministering a pharmaceutically effective amount of the pharmaceuticalcomposition according to claim
 8. 11. A method according to claim 10,wherein the infection is caused by Moraxella catarrhalis.
 12. A nucleicacid sequence encoding a peptide according to claim 1, or a homologue,polymorphism, fragment, degenerate or a splice variant thereof.
 13. Apolypeptide or a polypeptide truncate comprising at least one of theconserved sequences of Sequence ID No. 2 or Sequence ID No. 3, or afragment, homologue, derivative, degenerate, a hydroxylation,sulphonation or glycosylation product, or other secondary processingproduct thereof that retains the ability of binding fibronectin.
 14. Avaccine composition comprising UspA1 and/or UspA2, protein MID, and oneor more pharmaceutically acceptable adjuvants, vehicles, excipients,binders, carriers, or preservatives.
 15. A method for the treatment orprophylaxis of an infection in an individual comprising administering apharmaceutically effective amount of UspA1 and/or UspA2 and an effectiveamount of protein MID.
 16. A method according to claim 15, wherein theinfection is caused by Moraxella catarrhalis.
 17. A method for thetreatment or prophylaxis of an infection in an individual, comprisingadministering a pharmaceutically effective amount of a compositioncomprising a substance according to claim
 2. 18. A method according toclaim 17, wherein the infection is caused by Moraxella catarrhalis. 19.A method according to claim 17, wherein the infection is otitis media,sinusitis, or a lower respiratory tract infection.
 20. A vaccinecomposition comprising UspA1 and/or UspA2 and one or morepharmaceutically acceptable adjuvants, vehicles, excipients, binders,carriers, or preservatives.
 21. A fusion protein comprising one or morepeptides according to claim
 1. 22. A fusion protein comprising one ormore peptides according to claim
 2. 23. A nucleic acid sequence encodinga peptide according to claim 2, or a homologue, polymorphism, fragment,degenerate or a splice variant thereof.